Spray-dried composition comprising beta-galactosidase having transgalactosylating activity in combination with maltodextrin and/or nacl and application of the composition

ABSTRACT

A spray-dried composition comprising an enzyme which is a β-galactosidase having transgalactosylating activity and a maltodextrin and/or sodium chloride.

FIELD OF THE INVENTION

This invention relates to polypeptide-containing particles, to processesfor preparing polypeptide-containing particles, and to the use ofpeptide-containing particles.

BACKGROUND TO THE INVENTION

Galactooligosaccharides (GOS) are carbohydrates which are nondigestablein humans and animals comprising two or more galactose molecules,typically up to nine, linked by glycosidic bonds. GOS's may also includeone or more glucose molecules. One of the beneficial effects of GOS's istheir ability of acting as prebiotic compounds by selectivelystimulating the proliferation of beneficial colonic microorganisms suchas bacteria to give physiological benefits to the consumer. Theestablished health effects have resulted in a growing interest in GOSsas food ingredients for various types of food.

The enzyme β-galactosidase (EC 3.2.1.23) usually hydrolyses lactose tothe monosaccharides D-glucose and D-galactose. In the normal enzymereaction of β-galactosidases, the enzyme hydrolyses lactose andtransiently binds the galactose monosaccharide in a galactose-enzymecomplex that transfers galactose to the hydroxyl group of water,resulting in the liberation of D-galactose and D-glucose. However, athigh lactose concentrations some β-galactosidases are able to transfergalactose to the hydroxyl groups of D-galactose or D-glucose in aprocess called transgalactosylation whereby galacto-oligosaccharides areproduced. Also at high lactose concentrations some β-galactosidases areable to transfer galactose to the hydroxyl groups of lactose or higherorder oligosaccharides.

The genus Bifidobacterium is one of the most commonly used types ofbacteria cultures in the dairy industry for fermenting a variety ofdiary products. Ingestion of Bifidobacterium-containing productsfurthermore has a health-promoting effect. This effect is not onlyachieved by a lowered pH of the intestinal contents but also by theability of Bifidobacterium to repopulate the intestinal flora inindividuals who have had their intestinal flora disturbed by for exampleintake of antibiotics. Bifidobacterium furthermore has the potential ofoutcompeting potential harmful intestinal micro-organisms.

Galacto-oligosaccharides are known to enhance the growth ofBifidobacterium. This effect is likely achieved through the uniqueability of Bifidobacterium to exploit galacto-oligosaccharides as acarbon source. Dietary supplement of galacto-oligosaccharides isfurthermore thought to have a number of long-term disease protectingeffects. For example, galacto-oligosaccharide intake has been shown tobe highly protective against development of colorectal cancer in rats.There is therefore a great interest in developing cheap and efficientmethods for producing galacto-oligosaccharides for use in the industryfor improving dietary supplements and dairy products.

An extracellular lactase from Bifidobacterium bifidum DSM20215 truncatedwith approximately 580 amino acids (BIF3-d3) has been described as atransgalactosylating enzyme in a solution containing lactose solubilisedin water (Jorgensen et al. (2001), Appl. Microbiol. Biotechnol., 57:647-652). WO 01/90317 also describes a truncation variant (OLGA347) asbeing a transgalactosylating enzyme and in WO 2012/010597 OLGA347 wasshown to transfer a galactose moiety to D-fucose, N-acetyl-galactosamineand xylose.

In WO 2009/071539 a differently truncated fragment compared to BIF3-d3is described as resulting in efficient hydrolysis and very lowproduction of GOS when tested in milk.

In WO 2013/182686 we describe a polypeptide which has a useful ratio oftransgalactosylation to hydrolysis activity and thus is an efficientproducer of GOS when incubated with lactose even at low lactose levelssuch as in a milk-based product.

There is still a need however to provide improved formulations for suchenzymes. These products should provide the enzyme product with desiredproperties such as enzyme storage stability. The present inventionaddresses this need.

Surprising Features and Advantages of the Invention

Current commercial lactase products are formulated as liquids, e.g. inpolyols such as glycerol. It is believed that such formulations arephysically stable in that the enzyme is stable over an acceptable shelflife for the product.

We investigated formulating a polypeptide having transgalactosylatingactivity in glycerol. Using turbidity as a measure of precipitation, wefound that the physical stability of such a formulation was acceptable.

However, when we investigated the activity of such a formulation wefound that contrary to expectations, the presence of a polyol (e.g.,glycerol) perturbed the transgalactosylation activity of the enzyme. Inparticular, we found that in the presence of a polyol there was atendency for a galactosyl-glycerol to be generated instead of thedesired GOS. Surprisingly, we found that a spray-dried productcomprising a lactase in which a maltodextrin (or a combination ofmaltodextrins) or sodium chloride is used as a carrier is bothphysically stable and retains the transgalactosylation activity of theenzyme allowing for the synthesis of the desired GOS.

SUMMARY OF THE INVENTION

It is an aim of embodiments of the invention to provide a formulationfor a polypeptide which has a useful ratio of transgalactosylation tohydrolysis activity and in some embodiments where the polypeptide is anefficient producer of GOS when incubated with lactose even at lowlactose levels such as in a milk-based product.

According to one aspect of the present invention there is provided aspray-dried composition comprising a polypeptide which is aβ-galactosidase having transgalactosylating activity and a maltodextrin.

According to another aspect of the present invention there is provided aspray-dried composition comprising a polypeptide which is aβ-galactosidase having transgalactosylating activity and sodiumchloride.

According to another aspect of the present invention there is provided aspray-dried composition comprising a polypeptide which is aβ-galactosidase having transgalactosylating activity and maltodextrinand/or sodium chloride.

In one embodiment wherein the polypeptide is an enzyme which isclassified in Enzyme Classification (E.C.) 3.2.1.23.

In one embodiment the polypeptide has a ratio of transgalactosylatingactivity: β-galactosidase activity of at least 0.5, at least 1, at least2, at least 2.5, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, or at least12 at or above a concentration of 3% w/w initial lactose concentration.

In one embodiment the polypeptide having a transgalactosylating activityis selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 97% sequence identity with SEQ ID NO: 2, wherein said        polypeptide consists of at most 975 amino acid residues,    -   c. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   d. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5; or ii)        the complementary strand of i),    -   e. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   f. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In one embodiment the polypeptide having transgalactosylating activityis selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   c. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   d. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   e. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In one embodiment the polypeptide having transgalactosylating activitycomprises or consists of the amino acid sequence of SEQ ID NO:1, 2, 3, 4or 5.

In one embodiment the composition contain 0.1 wt % or less polyol.

In one embodiment the composition contains 0.1 wt % or less glycerol.

In one aspect, the particles of the spray dried composition have avolume mean diameter greater than 30 μm. In the present context, theparticle size of a powder may be measured as “volume mean diameter” suchas described by Rawle, A.: “Basic principles of particle size analysis”in Surface Coating International 2003, vol. 86, n° 2, pp. 58-65.Measurement of particle size in the work leading to this patent has beenperformed by laser diffraction (also known as Low Angle Laser LightScattering, or LALLS) using a particle size analyser model Mastersizer Sfrom company Malvern Ltd, UK.

In one embodiment the polypeptide having transgalactosylating activity,which comprises an amino acid sequence having at least 90% sequenceidentity with SEQ ID NO: 1, and wherein said polypeptide, when being anexpression product in a suitable host strain (e.g., Bacillus subtilis)comprising a nucleic acid sequence which encodes said polypeptide, isthe only polypeptide expression product of said nucleic acid sequencethat exhibits transgalactosylating activity.

In one embodiment the polypeptide is a C-terminal truncated fragment ofSEQ ID NO:22 having transgalactosylating activity and which is stableagainst further truncation such as by proteolytic degradation whenproduced in a suitable organism such as Bacillus subtilis and/or whichis stable against further truncation during storage after finalformulation.

In one embodiment the polypeptide comprises an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 1, wherein saidpolypeptide consists of at most 980 amino acid residues.

In one embodiment the polypeptide comprises an amino acid sequencehaving at least 97% sequence identity with SEQ ID NO: 2, wherein saidpolypeptide consists of at most 975 amino acid residues.

In one embodiment the polypeptide comprises an amino acid sequencehaving at least 96.5% sequence identity with SEQ ID NO: 3, wherein saidpolypeptide consists of at most 1300 amino acid residues.

In one aspect, disclosed herein is a composition comprising aspray-dried composition as described herein, preferably a foodcomposition, more preferably a dairy product.

In one aspect, disclosed herein is a method for producing a food productsuch as a dairy product by treating a milk-based substrate comprisinglactose with a spray-dried composition as described herein.

In one aspect, disclosed herein is a galacto-oligosaccharide orcomposition thereof obtained by treating a substrate comprising lactosewith a spray-dried composition as described herein. In one aspect,disclosed herein is a method of spray drying a liquid compositioncomprising:

-   -   (a) introducing a liquid composition into a spray drying        apparatus, wherein the liquid composition comprises an enzyme as        defined herein and a maltodextrin; and    -   (b) spray drying the liquid composition to produce particles.

In one aspect, disclosed herein is a method of spray drying a liquidcomposition comprising:

-   -   (a) introducing a liquid composition into a spray drying        apparatus, wherein the liquid composition comprises an enzyme as        defined herein and sodium chloride; and    -   (b) spray drying the liquid composition to produce particles.

LEGENDS TO THE FIGURE

FIG. 1 shows the results of Example 1.

FIGS. 2 and 3 show the results of Example 2.

FIG. 4 shows the results of Example 3.

FIG. 5 shows the results of Example 4.

SEQUENCE LISTING

SEQ ID NO: 1 (also named (BIF_917) herein) is a 887 amino acid truncatedfragment of SEQ ID NO: 22.

SEQ ID NO: 2 (also named (BIF_995) herein) is a 965 amino acid truncatedfragment of SEQ ID NO: 22.

SEQ ID NO: 3 (also named (BIF_1068) herein) is a 1038 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 4 (also named (BIF_1172) herein) is a 1142 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 5 (also named (BIF_1241) herein) is a 1211 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 6 (also named (BIF_1326) herein) is a 1296 amino acidtruncated fragment of SEQ ID NO: 22.

SEQ ID NO: 7 is Bifidobacterium bifidum glycoside hydrolase catalyticcore

SEQ ID NO: 8 is a nucleotide sequence encoding an extracellular lactasefrom Bifidobacterium bifidum DSM20215

SEQ ID NO: 9 is nucleotide sequence encoding BIF_917

SEQ ID NO: 10 is nucleotide sequence encoding BIF_995

SEQ ID NO: 11 is nucleotide sequence encoding BIF 1068

SEQ ID NO: 12 is nucleotide sequence encoding BIF_1172

SEQ ID NO: 13 is nucleotide sequence encoding BIF_1241

SEQ ID NO: 14 is nucleotide sequence encoding BIF_1326

SEQ ID NO: 15 is forward primer for generation of above BIF variants

SEQ ID NO: 16 is reverse primer for BIF917

SEQ ID NO: 17 is reverse primer for BIF995

SEQ ID NO: 18 is reverse primer for BIF1068

SEQ ID NO: 19 is reverse primer for BIF1241

SEQ ID NO: 20 is reverse primer for BIF1326

SEQ ID NO: 21 is reverse primer for BIF1478

SEQ ID NO: 22 is extracellular lactase from Bifidobacterium bifidumDSM20215

SEQ ID NO: 23 is signal sequence of extracellular lactase fromBifidobacterium bifidum DSM20215.

DETAILED DISCLOSURE OF THE INVENTION Definitions

In accordance with this detailed description, the followingabbreviations and definitions apply. It should be noted that as usedherein, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a polypeptide” includes a plurality of such polypeptides,and reference to “the formulation” includes reference to one or moreformulations and equivalents thereof known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The following terms are provided below.

“Transgalactosylase” means an enzyme that, among other things, is ableto transfer galactose to the hydroxyl groups of D-galactose or D-glucosewhereby galacto-oligosaccharides are produced. In one aspect, atransgalactosylase is identified by reaction of the enzyme on lactose inwhich the amount of galactose generated is less than the amount ofglucose generated at any given time.

In the present context, the term “transgalactosylating activity” meansthe transfer of a galactose moiety to a molecule other than water. Theactivity can be measured as [glucose]−[galactose] generated at any giventime during reaction or by direct quantification of the GOS generated atany given time during the reaction. This measurement may be performed inseveral ways such as by a HPLC method as shown in the examples. Whencomparing measurements of transgalactosylating activity, they have beenperformed at a given initial lactose concentration, such as e.g. 3, 4,5, 6, 7, 8, 9 or 10% (w/w).

In the present context, the term “β-galactosidase activity” means theability of an enzyme to hydrolyse β-galactosides such as for examplelactose into monosaccharides, glucose and galactose.

In the context of calculating transgalactosylating activity:β-galactosidase activity, the β-galactosidase activity is measured as[galactose] generated at any given time during reaction.

This measurement may be performed in several ways such as by a HPLCmethod as shown in the examples.

In the present context, the term “ratio of transgalactosylationactivity” using ortho-nitrophenol-β-D-galactopyranoside (ON PG) wascalculated as follows: Ratio is calculated as ratio between Abs420 withacceptor present divided by Abs420 without acceptor present times 100.Variant at or below index 100 are purely hydrolytic variants, whereasthe level above depicts relative transgalactosylating activity.

Ratio of transgalactosylationactivity=(Abs420^(+Cellobiose)/Abs420^(−Cellobiose))*100%, whereAbs420^(+Cellobiose) is the absorbance read at 420 nm using thedescribed method 3 below including cellobiose in the reaction andAbs420^(−Cellobiose) is the absorbance read at 420 nm using thedescribed method 3 below but without cellobiose in the reaction. Theequation above is only valid for dilutions where the absorbance isbetween 0.5 and 1.0.

In one aspect, the activity is measured after 15 min. reaction, 30 min.reaction, 60 min. reaction, 90 min. reaction, 120 min. reaction or 180min. reaction. Thus in one aspect, as an example the relativetransgalactosylation activity is measured 15 minutes after addition ofenzyme, such as 30 minutes after addition of enzyme, such as 60 minutesafter addition of enzyme, such as 90 minutes after addition of enzyme,such as 120 minutes after addition of enzyme or such as 180 minutesafter addition of enzyme.

In the present context, the term “ratio of transgalactosylatingactivity: β-galactosidase activity” means([Glucose]−[Galactose]/[Galactose]).

In the present context, the term [Glucose] means the glucoseconcentration in % by weight as measured by HPLC.

In the present context, the term [Galactose] means the galactoseconcentration in % by weight as measured by HPLC.

In the present context, the term “lactose has been transgalactosylated”means that a galactose molecule has been covalently linked to thelactose molecule such as for example covalently linked to any of thefree hydroxyl groups in the lactose molecule or as generated by internaltransgalactosylation for example forming allolactose.

In the present context, the evaluation of performance of polypeptidesdisclosed herein in galactooligosaccharide (GOS) production were testedin a “milk-based assay” (yogurt application mimic). Batch experimentswith a volume of 100 μl were performed in 96 well MTP plates using ayogurt mix, consisting of 98.60% (w/v) fresh pasteurized low-fat milk(Arla Mini-mælk) and 1.4% (w/v) Nutrilac YQ-5075 whey ingredient (Arla).To completely hydrate Nutrilac YQ-5075 the mixture was left withagitation for 20 h and afterwards added 20 mM NaPhosphate pH 6.5 toensure a pH of 6.5. This yogurt-base was either used plain or withvarious supplements such as additional lactose, fucose, maltose, xyloseor salts. 90 μl of the yogurt was mixed with 10 μl purified enzyme orcrude ferment, sealed with tape and incubated at 43° C. for 3 hours. Thereaction was stopped by 100 μl 10% Na2CO3. Samples were stored at −20°C. Quantification of galactooligosaccharides (GOS), lactose, glucose andgalactose were performed by HPLC. Analysis of samples was carried out ona Dionex ICS 3000. IC parameters were as follows: Mobile phase: 150 mMNaOH, Flow: Isochratic, 0.25 ml/min, Column: Carbopac PA1, Columntemperature: RT, Injection volume: 10 μL, Detector: PAD, Integration:Manual, Sample preparation: 100 times dilution in Milli-Q water (0.1 mlsample+9.9 ml water) and filtration through 0.45 ìm syringe filters,Quantification: Peak areas in percent of peak area of the standard. AGOS syrup (Vivanal GOS, Friesland Campina) was used as standard for GOSquantification. Results of such an evaluation is shown in FIG. 4, andfurther described in example 2.

In the present context, the term “which polypeptide is spray-dried”means that the polypeptide has been obtained by spray-drying apolypeptide which is in solution or suspension at an appropriatetemperature and for an appropriate period removing the water.

In the present context, the term “which polypeptide is in solution”relates to a polypeptide which is soluble in a solvent withoutprecipitating out of solution. A solvent for this purpose includes anymillieu in which the polypeptide may occur, such as an aqueous buffer orsalt solution, a fermentation broth, or the cytoplasm of an expressionhost.

In the present context, the term “stabilizer” means any stabilizer forstabilizing the polypeptide e.g., a polyol such as, e.g., glycerol orpropylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid,or a boric acid derivative (e.g., an aromatic borate ester). In oneaspect, the stabilizer is not a polyol, or the polyol is present at alevel of 0.1 wt % or less.

The term “isolated” means that the polypeptide is at least substantiallyfree from at least one other component with which the sequence isnaturally associated in nature and as found in nature. In one aspect,“isolated polypeptide” as used herein refers to a polypeptide which isat least 30% pure, at least 40% pure, at least 60% pure, at least 80%pure, at least 90% pure, and at least 95% pure, as determined bySDS-PAGE.

The term “substantially pure polypeptide” means herein a polypeptidepreparation which contains at most 10%, preferably at most 8%, morepreferably at most 6%, more preferably at most 5%, more preferably atmost 4%, at most 3%, even more preferably at most 2%, most preferably atmost 1%, and even most preferably at most 0.5% by weight of otherpolypeptide material with which it is natively associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polypeptides are in “essentiallypure form”, i.e., that the polypeptide preparation is essentially freeof other polypeptide material with which it is natively associated. Thiscan be accomplished, for example, by preparing the polypeptide by meansof well-known recombinant methods or by classical purification methods.Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

The term “purified” or “pure” means that a given component is present ata high level state—e.g. at least about 51% pure, such as at least 51%pure, or at least about 75% pure such as at least 75% pure, or at leastabout 80% pure such as at least 80% pure, or at least about 90% puresuch as at least 90% pure, or at least about 95% pure such as at least95% pure, or at least about 98% pure such as at least 98% pure. Thecomponent is desirably the predominant active component present in acomposition.

The term “microorganism” in relation to the present invention includesany “microorganism” that could comprise a nucleotide sequence accordingto the present invention or a nucleotide sequence encoding for apolypeptide having the specific properties as defined herein and/orproducts obtained therefrom. In the present context, “microorganism” mayinclude any bacterium or fungus being able to ferment a milk substrate.

The term “host cell”—in relation to the present invention includes anycell that comprises either a nucleotide sequence encoding a polypeptidehaving the specific properties as defined herein or an expression vectoras described above and which is used in the production of a polypeptidehaving the specific properties as defined herein. In one aspect, theproduction is recombinant production.

The term “milk”, in the context of the present invention, is to beunderstood as the lacteal secretion obtained from any mammal, such ascows, sheep, goats, buffaloes or camels.

In the present context, the term “milk-based substrate” means any rawand/or processed milk material or a material derived from milkconstituents. Useful milk-based substrates include, but are not limitedto solutions/suspensions of any milk or milk like products comprisinglactose, such as whole or low fat milk, skim milk, buttermilk,reconstituted milk powder, condensed milk, solutions of dried milk, UHTmilk, whey, whey permeate, acid whey, or cream. Preferably, themilk-based substrate is milk or an aqueous solution of skim milk powder.The milk-based substrate may be more concentrated than raw milk. In oneembodiment, the milk-based substrate has a ratio of protein to lactoseof at least 0.2, preferably at least 0.3, at least 0.4, at least 0.5, atleast 0.6 or, most preferably, at least 0.7. The milk-based substratemay be homogenized and/or pasteurized according to methods known in theart.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. It may be performed so as to break up the milkfat into smaller sizes so that it no longer separates from the milk.This may be accomplished by forcing the milk at high pressure throughsmall orifices.

“Pasteurizing” as used herein means reducing or eliminating the presenceof live organisms, such as microorganisms, in the milk-based substrate.Preferably, pasteurization is attained by maintaining a specifiedtemperature for a specified period of time. The specified temperature isusually attained by heating. The temperature and duration may beselected in order to kill or inactivate certain bacteria, such asharmful bacteria, and/or to inactivate enzymes in the milk. A rapidcooling step may follow. A “food product” or “food composition” in thecontext of the present invention may be any comestible food or feedproduct suitable for consumption by an animal or human.

A “dairy product” in the context of the present invention may be anyfood product wherein one of the major constituents is milk-based.Preferable, the major constituent is milk-based. More preferably, themajor constituent is a milk-based substrate which has been treated withan enzyme having transgalactosylating activity.

Maltodextrin is a polysaccharide that can be used as a food additive.Maltodextrin consists of D-glucose units connected in chains of variablelength. The glucose units are primarily linked with α(1→4) glycosidicbonds. Maltodextrin is typically composed of a mixture of chains thatvary from three to seventeen glucose units long.

Maltodextrins are classified by DE (dextrose equivalent) and have a DEbetween 3 to 20. The higher the DE value, the shorter the glucosechains, the higher the sweetness, the higher the solubility and thelower heat resistance. Above DE 20, the European Union's CN code callsit glucose syrup, at DE 10 or lower the customs CN code nomenclatureclassifies maltodextrins as dextrins. The present invention may employ amixture of such maltodextrins.

In the present context, “one of the major constituents” means aconstituent having a dry matter which constitutes more than 20%,preferably more than 30% or more than 40% of the total dry matter of thedairy product, whereas “the major constituent” means a constituenthaving a dry matter which constitutes more than 50%, preferably morethan 60% or more than 70% of the total dry matter of the dairy product.

A “fermented dairy product” in present context is to be understood asany dairy product wherein any type of fermentation forms part of theproduction process. Examples of fermented dairy products are productslike yoghurt, buttermilk, creme fraiche, quark and fromage frais.Another example of a fermented dairy product is cheese. A fermenteddairy product may be produced by any method known in the art.

The term “fermentation” means the conversion of carbohydrates intoalcohols or acids through the action of a microorganism such as astarter culture. In one aspect, fermentation comprises conversion oflactose to lactic acid.

In the present context, “microorganism” may include any bacterium orfungus being able to ferment a milk substrate.

In the present context the term “Pfam domains” means regions within aprotein sequence that are identified as either Pfam-A or Pfam-B based onmultiple sequence alignments and the presence of Hidden Markov Motifs(“The Pfam protein families database”: R. D. Finn, J. Mistry, J. Tate,P. Coggill, A. Heger, J. E. Pollington, O. L. Gavin, P. Gunesekaran, G.Ceric, K. Forslund, L. Holm, E. L. Sonnhammer, S. R. Eddy, A. BatemanNucleic Acids Research (2010) Database Issue 38:D211-222.). As examplesof Pfam domains mention may be made of Glyco_hydro2N (PF02837),Glyco_hydro (PF00703), Glyco_hydro 2C (PF02836) and Bacterial Ig-likedomain (group 4) (PF07532).

As used herein “a position corresponding to position” means that analignment as described herein is made between a particular querypolypeptide and the reference polypeptide. The position corresponding toa specific position in the reference polypeptide is then identified asthe corresponding amino acid in the alignment with the highest sequenceidentity.

A “variant” or “variants” refers to either polypeptides or nucleicacids. The term “variant” may be used interchangeably with the term“mutant”. Variants include insertions, substitutions, transversions,truncations, and/or inversions at one or more locations in the aminoacid or nucleotide sequence, respectively. The phrases “variantpolypeptide”, “polypeptide variant”, “polypeptide”, “variant” and“variant enzyme” mean a polypeptide/protein that has an amino acidsequence that either has or comprises a selected amino acid sequence ofor is modified compared to the selected amino acid sequence, such as SEQID NO: 1, 2, 3, 4 or 5.

As used herein, “reference enzymes,” “reference sequence,” “referencepolypeptide” mean enzymes and polypeptides from which any of the variantpolypeptides are based, e.g., SEQ ID NO: 1, 2, 3, 4 or 5. A “referencenucleic acid” means a nucleic acid sequence encoding the referencepolypeptide.

As used herein, the terms “reference sequence” and “subject sequence”are used interchangeably.

As used herein, “query sequence” means a foreign sequence, which isaligned with a reference sequence in order to see if it falls within thescope of the present invention. Accordingly, such query sequence can forexample be a prior art sequence or a third party sequence.

As used herein, the term “sequence” can either be referring to apolypeptide sequence or a nucleic acid sequence, depending of thecontext.

As used herein, the terms “polypeptide sequence” and “amino acidsequence” are used interchangeably.

The signal sequence of a “variant” may be the same or may differ fromthe signal sequence of the wild-type a Bacillus signal peptide or anysignal sequence that will secrete the polypeptide. A variant may beexpressed as a fusion protein containing a heterologous polypeptide. Forexample, the variant can comprise a signal peptide of another protein ora sequence designed to aid identification or purification of theexpressed fusion protein, such as a His-Tag sequence.

To describe the various variants that are contemplated to be encompassedby the present disclosure, the following nomenclature will be adoptedfor ease of reference. Where the substitution includes a number and aletter, e.g., 592P, then this refers to {position according to thenumbering system/substituted amino acid}. Accordingly, for example, thesubstitution of an amino acid to proline in position 592 is designatedas 592P. Where the substitution includes a letter, a number, and aletter, e.g., D592P, then this refers to {original amino acid/positionaccording to the numbering system/substituted amino acid}.

Accordingly, for example, the substitution of alanine with proline inposition 592 is designated as A592P.

Where two or more substitutions are possible at a particular position,this will be designated by contiguous letters, which may optionally beseparated by slash marks “/”, e.g., G303ED or G303E/D.

Position(s) and substitutions are listed with reference to for exampleeither SEQ ID NO: 1, 2, 3, 4 or 5. For example equivalent positions inanother sequence may be found by aligning this sequence with either SEQID NO: 1, 2, 3, 4 or 5 to find an alignment with the highest percentidentity and thereafter determining which amino acid aligns tocorrespond with an amino acid of a specific position of either SEQ IDNO: 1, 2, 3, 4 or 5. Such alignment and use of one sequence as a firstreference is simply a matter of routine for one of ordinary skill in theart.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

As used herein, “polypeptide” is used interchangeably with the terms“amino acid sequence”, “enzyme”, “peptide” and/or “protein”. As usedherein, “nucleotide sequence” or “nucleic acid sequence” refers to anoligonucleotide sequence or polynucleotide sequence and variants,homologues, fragments and derivatives thereof. The nucleotide sequencemay be of genomic, synthetic or recombinant origin and may bedouble-stranded or single-stranded, whether representing the sense oranti-sense strand. As used herein, the term “nucleotide sequence”includes genomic DNA, cDNA, synthetic DNA, and RNA.

“Homologue” means an entity having a certain degree of identity or“homology” with the subject amino acid sequences and the subjectnucleotide sequences. In one aspect, the subject amino acid sequence isSEQ ID NO: 1, 2, 3, 4 or 5, and the subject nucleotide sequencepreferably is SEQ ID NO: 9, 10, 11, 12 or 13.

A “homologous sequence” includes a polynucleotide or a polypeptidehaving a certain percent, e.g., 80%, 85%, 90%, 95%, or 99%, of sequenceidentity with another sequence. Percent identity means that, whenaligned, that percentage of bases or amino acid residues are the samewhen comparing the two sequences. Amino acid sequences are notidentical, where an amino acid is substituted, deleted, or addedcompared to the subject sequence. The percent sequence identitytypically is measured with respect to the mature sequence of the subjectprotein, i.e., following removal of a signal sequence, for example.Typically, homologues will comprise the same active site residues as thesubject amino acid sequence. Homologues also retain enzymatic activity,although the homologue may have different enzymatic properties than thewild-type.

As used herein, “hybridization” includes the process by which a strandof nucleic acid joins with a complementary strand through base pairing,as well as the process of amplification as carried out in polymerasechain reaction (PCR) technologies. The variant nucleic acid may exist assingle- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or anRNA/DNA copolymer. As used herein, “copolymer” refers to a singlenucleic acid strand that comprises both ribonucleotides anddeoxyribonucleotides. The variant nucleic acid may be codon-optimized tofurther increase expression.

As used herein, a “synthetic” compound is produced by in vitro chemicalor enzymatic synthesis. It includes, but is not limited to, variantnucleic acids made with optimal codon usage for host organisms, such asa yeast cell host or other expression hosts of choice.

As used herein, “transformed cell” includes cells, including bothbacterial and fungal cells, which have been transformed by use ofrecombinant DNA techniques. Transformation typically occurs by insertionof one or more nucleotide sequences into a cell. The inserted nucleotidesequence may be a heterologous nucleotide sequence, i.e., is a sequencethat is not natural to the cell that is to be transformed, such as afusion protein.

As used herein, “operably linked” means that the described componentsare in a relationship permitting them to function in their intendedmanner. For example, a regulatory sequence operably linked to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under condition compatible with the control sequences.

As used herein, the term “fragment” is defined herein as a polypeptidehaving one or more (several) amino acids deleted from the amino and/orcarboxyl terminus wherein the fragment has activity.

In one aspect, the term “fragment” is defined herein as a polypeptidehaving one or more (several) amino acids deleted from the amino and/orcarboxyl terminus of the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5;wherein the fragment has transgalactosylating activity.

The term “Galactose Binding domain-like” as used herein is abbreviatedto and interchangeable with the term “GBD”.

Degree of Identity

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

In one embodiment, the degree of sequence identity between a querysequence and a reference sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the length of the reference sequence.

In one embodiment, the degree of sequence identity between a querysequence and a reference sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the length of the longest of the twosequences.

In another embodiment, the degree of sequence identity between the querysequence and the reference sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the “alignment length”, where the alignmentlength is the length of the entire alignment including gaps andoverhanging parts of the sequences.

Sequence identity comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs use complex comparisonalgorithms to align two or more sequences that best reflect theevolutionary events that might have led to the difference(s) between thetwo or more sequences. Therefore, these algorithms operate with ascoring system rewarding alignment of identical or similar amino acidsand penalising the insertion of gaps, gap extensions and alignment ofnon-similar amino acids. The scoring system of the comparison algorithmsinclude:

-   -   i) assignment of a penalty score each time a gap is inserted        (gap penalty score),    -   ii) assignment of a penalty score each time an existing gap is        extended with an extra position (extension penalty score),    -   iii) assignment of high scores upon alignment of identical amino        acids, and    -   iv) assignment of variable scores upon alignment of        non-identical amino acids.

Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons.

The scores given for alignment of non-identical amino acids are assignedaccording to a scoring matrix also called a substitution matrix. Thescores provided in such substitution matrices are reflecting the factthat the likelihood of one amino acid being substituted with anotherduring evolution varies and depends on the physical/chemical nature ofthe amino acid to be substituted. For example, the likelihood of a polaramino acid being substituted with another polar amino acid is highercompared to being substituted with a hydrophobic amino acid. Therefore,the scoring matrix will assign the highest score for identical aminoacids, lower score for non-identical but similar amino acids and evenlower score for non-identical non-similar amino acids. The mostfrequently used scoring matrices are the PAM matrices (Dayhoff et al.(1978), Jones et al. (1992)), the BLOSUM matrices (Henikoff and Henikoff(1992)) and the Gonnet matrix (Gonnet et al. (1992)).

Suitable computer programs for carrying out such an alignment include,but are not limited to, Vector NTI (Invitrogen Corp.) and the ClustalV,ClustalW and ClustalW2 programs (Higgins D G & Sharp P M (1988), Higginset al. (1992), Thompson et al. (1994), Larkin et al. (2007). A selectionof different alignment tools is available from the ExPASy Proteomicsserver at www.expasy.org. Another example of software that can performsequence alignment is BLAST (Basic Local Alignment Search Tool), whichis available from the webpage of National Center for BiotechnologyInformation which can currently be found at http://www.ncbi.nlm.nih.gov/and which was firstly described in Altschul et al. (1990) J. Mol. Biol.215; 403-410.

In a preferred embodiment of the present invention, the alignmentprogram is performing a global alignment program, which optimizes thealignment over the full-length of the sequences. In a further preferredembodiment, the global alignment program is based on theNeedleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D.(1970), “A general method applicable to the search for similarities inthe amino acid sequence of two proteins”, Journal of Molecular Biology48 (3): 443-53). Examples of current programs performing globalalignments using the Needleman-Wunsch algorithm are EMBOSS Needle andEMBOSS Stretcher programs, which are both available athttp://www.ebi.ac.uk/Tools/psa/.

EMBOSS Needle performs an optimal global sequence alignment using theNeedleman-Wunsch alignment algorithm to find the optimum alignment(including gaps) of two sequences along their entire length.

EMBOSS Stretcher uses a modification of the Needleman-Wunsch algorithmthat allows larger sequences to be globally aligned.

In one embodiment, the sequences are aligned by a global alignmentprogram and the sequence identity is calculated by identifying thenumber of exact matches identified by the program divided by the“alignment length”, where the alignment length is the length of theentire alignment including gaps and overhanging parts of the sequences.

In a further embodiment, the global alignment program uses theNeedleman-Wunsch algorithm and the sequence identity is calculated byidentifying the number of exact matches identified by the programdivided by the “alignment length”, where the alignment length is thelength of the entire alignment including gaps and overhanging parts ofthe sequences.

In yet a further embodiment, the global alignment program is selectedfrom the group consisting of EMBOSS Needle and EMBOSS stretcher and thesequence identity is calculated by identifying the number of exactmatches identified by the program divided by the “alignment length”,where the alignment length is the length of the entire alignmentincluding gaps and overhanging parts of the sequences.

Once the software has produced an alignment, it is possible to calculate% similarity and % sequence identity. The software typically does thisas part of the sequence comparison and generates a numerical result.

In one embodiment, it is preferred to use the ClustalW software forperforming sequence alignments. Preferably, alignment with ClustalW isperformed with the following parameters for pairwise alignment:

Substitution matrix: Gonnet 250 Gap open penalty: 20 Gap extensionpenalty: 0.2 Gap end penalty: None

ClustalW2 is for example made available on the internet by the EuropeanBioinformatics Institute at the EMBL-EBI webpage www.ebi.ac.uk undertools—sequence analysis—ClustalW2. Currently, the exact address of theClustalW2 tool is www.ebi.ac.uk/Tools/clustalw2.

In another embodiment, it is preferred to use the program Align X inVector NTI (Invitrogen) for performing sequence alignments. In oneembodiment, Exp10 has been may be used with default settings:

Gap opening penalty: 10

Gap extension penalty: 0.05

Gap separation penalty range: 8

In a another embodiment, the alignment of one amino acid sequence with,or to, another amino acid sequence is determined by the use of the scorematrix: blosum62mt2 and the VectorNTI Pair wise alignment settings

Settings K-tuple 1 Number of best diagonals 5 Window size 5 Gap Penalty3 Gap opening Penalty 10 Gap extension Penalty 0.1

In one embodiment, the percentage of identity of one amino acid sequencewith, or to, another amino acid sequence is determined by the use ofBlast with a word size of 3 and with BLOSUM 62 as the substitutionmatrix.

Polypeptides

In one aspect, the invention disclosed herein employs a polypeptidehaving a ratio of transgalactosylating activity: β-galactosidaseactivity of at least 0.5, at least 1, at least 2, at least 2.5, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, or at least 12 at or above a concentrationof 3% w/w initial lactose concentration.

In one aspect, the invention disclosed herein employs a polypeptide,wherein the glycoside hydrolase catalytic core has an amino acidsequence of SEQ ID NO:7.

In one aspect, the invention disclosed herein employs a polypeptidecontaining a Glyco_hydro2N (PF02837), a Glyco_hydro (PF00703) and/or aGlyco_hydro 2C (PF02836) domains.

In one aspect, disclosed herein is a polypeptide containing theBacterial Ig-like domain (group 4) (PF07532).

In one aspect, disclosed herein is a polypeptide havingtransgalactosylating activity selected from the group consisting of:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 97% sequence identity with SEQ ID NO: 2, wherein said        polypeptide consists of at most 975 amino acid residues,    -   c. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   d. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5; or ii)        the complementary strand of i),    -   e. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   f. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.

In another aspect, the invention disclosed herein employs a polypeptidehaving transgalactosylating activity selected from the group consistingof:

-   -   a. a polypeptide comprising an amino acid sequence having at        least 96.5% sequence identity with SEQ ID NO: 3, wherein said        polypeptide consists of at most 1300 amino acid residues,    -   b. a polypeptide comprising an amino acid sequence having at        least 90% sequence identity with SEQ ID NO: 1, wherein said        polypeptide consists of at most 980 amino acid residues,    -   c. a polypeptide encoded by a polynucleotide that hybridizes        under at least low stringency conditions with i) the nucleic        acid sequence comprised in SEQ ID NO: 9, 10, 11, 12 or 13        encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or ii)        the complementary strand of i),    -   d. a polypeptide encoded by a polynucleotide comprising a        nucleotide sequence having at least 70% identity to the        nucleotide sequence encoding for the polypeptide of SEQ ID NO:        1, 2, 3, 4 or 5 or the nucleotide sequence comprised in SEQ ID        NO: 9, 10, 11, 12 or 13 encoding a mature polypeptide, and    -   e. a polypeptide comprising a deletion, insertion and/or        conservative substitution of one or more amino acid residues of        SEQ ID NO: 1, 2, 3, 4 or 5.    -   f.

In one aspect, of the invention disclosed herein employs a polypeptide,wherein the amino acid sequence has at least 68%, 70%, 72%, 74%, 76%,78%, 80%%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,sequence identity to the mature amino acid sequence of SEQ ID NO: 1, 2,3, 4 or 5.

In one aspect, of the invention disclosed herein employs a polypeptidehaving 90% sequence identity to the mature amino acid sequence of SEQ IDNO:1.

In one aspect, of the invention disclosed herein employs a polypeptidehaving 90% sequence identity to the mature amino acid sequence of SEQ IDNO:2.

In one aspect, of the invention disclosed herein employs a polypeptidehaving 96.5% sequence identity to the mature amino acid sequence of SEQID NO:3.

In one aspect, of the invention disclosed herein employs a polypeptidehaving 96.5% sequence identity to the mature amino acid sequence of SEQID NO:4.

In one aspect, of the invention disclosed herein employs a polypeptidehaving 96.5% sequence identity to the mature amino acid sequence of SEQID NO:5.

In one aspect, of the invention disclosed herein employs a polypeptidecomprising or consisting of the amino acid sequence of SEQ ID NO:1, 2,3, 4 or 5.

In one aspect, of the invention disclosed herein employs a polypeptide,which is derived from Bifidobacterium bifidum.

In one aspect, of the invention disclosed herein employs a polypeptidehaving a pH optimum of 6.5-7.5.

In one aspect, of the invention disclosed herein employs a polypeptidehaving a temperature optimum of 30-60 such as 42-60 degree Celsius.

Polypeptides having activity on carbohydrates can be classified usingeither the IUBMB system of classification based on their substratespecificity or on the CaZy assignment into one of the current 125glycoside hydrolase family. In the CaZy database the assignment is basedon both sequence and structural information combined with knowledge ofstereochemistry of the substrates and products.

Disclosed herein is the use of polypeptides which when being anexpression product in a suitable host strain (e.g., Bacillus subtilis)comprising a nucleic acid sequence which encodes said polypeptide, isthe only polypeptide expression product of said nucleic acid sequencethat exhibits transgalactosylating activity. This may be evaluated byusing the following techniques know to a person skilled in the art. Thesamples to be evaluated are subjected to SDS-PAGE and visualized using adye appropriate for protein quantification, such as for example theBio-Rad Criterion system. The gel is then scanned using appropriatedensiometic scanner such as for example the Bio-Rad Criterion system andthe resulting picture is ensured to be in the dynamic range. The bandscorresponding to any variant/fragment derived from SEQ ID NO: 8 arequantified and the percentage of the polypeptides are calculated as:Percentage of polypeptide in question=polypeptide in question/(sum ofall polypeptides exhibiting transgalactosylating activity)*100. Thetotal number of polypeptides variants/fragments derived from SEQ ID NO:8in the composition can be determined by detecting fragment derived fromSEQ ID NO:8 by western blotting using a polyclonal antibody by methodsknow to a person skilled in the art.

The polypeptide disclosed herein comprises at least two separatefunctional domains contained within the enzyme. Firstly, the polypeptideshould contain a glycoside hydrolase catalytic core as described in thefollowing. The catalytic core should belong to the GH-A clan of relatedglycoside hydrolase families. The GH-A clan is characterized by cleavingglycosidic bonds via a retaining mechanism and possesses a catalyticdomain which is based on a TIM barrel fold (Werenga, 2001, FEBS Letters,492(3), p 193-8). The catalytic domain contains two glutamic acidresidues which act as proton donor and nucleophile, emanating fromstrands 4 and 7 of the barrel domain (Jenkins, 1995, FEBS Letters,362(3), p 281-5). The overall structure of the TIM barrel is a (β/α) 8fold consisting of 8 beta strands and 8 alpha-helices. In one aspect,the glycoside hydrolase catalytic core disclosed herein belong to eitherof the glycoside hydrolase families GH-2, and -35 which are allTIM-barrel enzymes belonging to the GH-A clan. In a further aspect, theglycoside hydrolase catalytic core belong to family GH-2 or GH-35. In afurther aspect, the glycoside hydrolase catalytic core belong to familyGH-2. A common denominator is that these enzymes are so called retainingenzymes, so that the stereochemistry of the substrate is conserved inthe product (Henrissat, 1997, Curr Opin Struct Biol, 7(5), 637-44).

In one aspect, the polypeptides disclosed herein have activity oncarbohydrates bonds which has the β(1→4) conformation. This effectivelyput the enzymes into the IUBMB EC 3.2.1.23 class of β-galactosidases.This activity may be, but is not confined to, determined by utilizingsynthetic substrates such as para-nitrophenol-β-D-galactopyranoside(PNPG), ortho-nitrophenol-β-D-galactopyranoside (ONPG) orβ-D-galactopyranoside with chromogenic aglycons (XGal). As analternative way of determining whether an enzyme belong to the EC3.2.1.23 class of β-galactosidases is to incubate with a substrate suchas lactose and measure the release of glucose by a method such asenzymatic determination, HPLC, TLC or other methods known to personsskilled in the art.

In order to predict functional entities of polypeptides severalavailable public repositories can be applied such as for example Pfam(Nucl. Acids Res. (2010) 38 (suppl 1): D211-D222. doi:10.1093/nar/gkp985) and Interpro (Nucl. Acids Res. (2009) 37 (suppl 1):D211-D215. doi: 10.1093/nar/gkn785). It should be specified that whenperforming such analysis the analysis should be performed on the fulllength sequence of the polypeptide available from public repositorydatabases.

In a further aspect, a polypeptide containing one or more Pfam domainsselected from: Glyco_hydro2N (PF02837), Glyco_hydro (PF00703),Glyco_hydro 2C (PF02836) and Bacterial Ig-like domain (group 4)(PF07532), is provided. In yet a further aspect, a polypeptidecontaining the Pfam domains Glyco_hydro2N (PF02837), Glyco_hydro(PF00703), Glyco_hydro 2C (PF02836) and Bacterial Ig-like domain (group4) (PF07532), is provided. In yet a further aspect, a polypeptidecontaining the Glyco_hydro2N (PF02837), Glyco_hydro (PF00703), andGlyco_hydro 2C (PF02836) domains which constitutes the catalytic domainof the polypeptide, is used.

In a further aspect, the polypeptide is derived from Bifidobacteriumbifidum.

In a further aspect, a polypeptide as disclosed herein and having aratio of transgalactosylating activity: β-galactosidase activity of atleast 1, at least 2.5, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, or atleast 12 as measured at a concentration of 100 ppm in a milk-based assayat 37° C. and 5 w/w % lactose after 15, 30 or 180 such as 180 minutesreaction, is used.

In one aspect, the herein disclosed polypeptide(s) has atransgalactosylating activity such that more than 20%, more than 30%,more than 40%, up to 50% of the initial lactose is transgalactosylatedas measured at a concentration of 100 ppm in a milk-based assay at 37°C. and 5 w/w % lactose after 15, 30 or 180 such as 180 minutes ofreaction. In a preferred embodiment of the invention the afore-mentionedtransgalactosylating activity is retained in the spray-dried compositionof the invention. In one embodiment the transgalactosylating activity isretained in the spray-dried composition through the storage period ofthe spray-dried composition. This storage period may be at least 1, atleast 2, at least 3, at least 4, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 18, or at least24 months.

In a further aspect, the herein disclosed polypeptide(s) has aβ-galactosidase activity such that less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20% ofthe lactose has been hydrolysed as measured at a concentration of 100ppm in a milk-based assay at 37° C. and 5 w/w % lactose after 15, 30 or180 such as 180 minutes of reaction. In a preferred embodiment of theinvention the afore-mentioned β-galactosidase activity is retained inthe spray-dried composition of the invention. In one embodiment theβ-galactosidase activity is retained in the spray-dried compositionthrough the storage period of the spray-dried composition. This storageperiod may be at least 1, at least 2, at least 3, at least 4, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 18, or at least 24 months.

In one aspect, the β-galactosidase activity and/or thetransgalactosylating activity are measured at a concentration of 100 ppmcorresponding to 2.13 LAU as specified in method 4. In general terms theunits of activity of the enzyme may be measured according to the assaydisclosed in WO 2003/186286 as Method 4 and reproduced herein as method4.

In a further aspect, the herein disclosed polypeptide(s) has one or moreof the following characteristics:

a) a ratio of transgalactosylating activity: β-galactosidase activity ofat least of at least 1, at least 2.5, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, or at least 12 as measured at a concentration of 100 ppm in amilk-based assay at 37° C. and 5 w/w % lactose after 15, 30 or 180 suchas 180 minutes reaction, and/or b) has a transgalactosylating activitysuch that more than 20%, more than 30%, more than 40%, and up to 50% ofthe initial lactose has been transgalactosylated as measured at aconcentration of 100 ppm in a milk-based assay at 37° C. and 5 w/w %lactose after 15, 30 or 180 such as 180 minutes of reaction.

In one aspect, a polypeptide comprising an amino acid sequence having atleast 96.5% sequence identity with SEQ ID NO: 3, wherein saidpolypeptide consists of at most 1300 amino acid residues, is provided.In a further aspect, a polypeptide comprising an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 1 such as whereinsaid sequence identity is at least 95%, such as, e.g. at least 96%, atleast 97%, at least 98%, at least 99% or at least 100% sequenceidentity, and wherein said polypeptide consists of at most 980 aminoacid residues, is provided. In a further aspect, a polypeptidecomprising an amino acid sequence having at least 90% sequence identitywith SEQ ID NO: 1, wherein said polypeptide consists of at most 980amino acid residues, is provided. In yet a further aspect, a polypeptidewherein said polypeptide has at least 90% sequence identity with SEQ IDNO: 1, such as wherein said polypeptide has at least 90%, such as, e.g.at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identitywith SEQ ID NO: 1 is provided. In another aspect, a polypeptide havingat least 96.5% sequence identity to SEQ ID NO: 2 such as wherein saidpolypeptide has at least 97%, such as, e.g. at least 98% or at least 99%sequence identity with SEQ ID NO: 2. In one aspect, the polypeptidesdisclosed herein consist of at the most 975 amino acid residues, suchas, e.g. at most 970 amino acid residues, such as at most 950 amino acidresidues, such as at most 940 amino acid residues, at most 930 aminoacid residues, at most 920 amino acid residues, at most 910 amino acidresidues, at most 900 amino acid residues, at most 895 amino acidresidues or at most 890 amino acid residues, is provided. In one aspect,a particular polypeptide consists of 887 or 965 amino acid residues, isprovided. In one aspect, a polypeptide comprising an amino acid sequencehaving at least 97% sequence identity with SEQ ID NO: 2 such as whereinsaid sequence identity is at least 98%, such as, e.g. at least 99% or atleast 100% sequence identity, wherein said polypeptide consists of atmost 975 amino acid residues, such as, e.g. at most 970 or at least 965amino acid residues, is provided. In one aspect, a polypeptidecomprising an amino acid sequence having at least 97% sequence identitywith SEQ ID NO: 2, wherein said polypeptide consists of at most 975amino acid residues, is used.

In a further preferred aspect, a polypeptide which comprises SEQ IDNO:1, 2, 3, 4 or 5, is provided. In yet a preferred aspect, apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3,4, or 5, especially a polypeptide consisting of the amino acid sequenceof SEQ ID NO: 1 or 2, is used.

In a further aspect, a polypeptide comprising an amino acid sequencehaving at least 96.5% sequence identity with SEQ ID NO: 3 such aswherein said sequence identity is at least 97%, such as, e.g. at least98%, at least 99% or at least 100% sequence identity, wherein saidpolypeptide consists of at most 1300 amino acid residues, is used.

In a further aspect, a polypeptide wherein said polypeptide has at least98.5%, such as at least 99% or at least 99.5% sequence identity with SEQID NO: 5, is provided. In one aspect, such a polypeptide consists of atmost 1290 amino acid residues, such as, e.g. at most 1280, at most 1270,at most 1260, at most 1250, at most 1240, at most 1230, at most 1220 orat most 1215 amino acid residues, is provided. In a preferred aspect, apolypeptide which consists of 1211 amino acid residues, is used.

In a further aspect, a polypeptide wherein said polypeptide has at least96% such as at least at least 97%, such as, e.g., at least 98% or atleast 99% sequence identity with SEQ ID NO: 4, is provided. In oneaspect, a polypeptide which consists of at most 1210 amino acidresidues, such as, e.g. at most 1200, at most 1190, at most 1180, atmost 1170, at most 1160, at most 1150 or at most 1145 amino acidresidues, such as 1142 amino acid residues, is used.

In a further aspect, a polypeptide wherein said polypeptide has at least96.5% such as at least 97%, such as, e.g., at least 98% or at least 99%sequence identity with SEQ ID NO: 3, is provided. In one aspect, apolypeptide which consists of at most 1130 amino acid residues, such as,e.g. at the most 1120, at the most 1110, at the most 1100, at the most1090, at the most 1080, at the most 1070, at the most 1060, at the most1050, at the most 1055 or at the most 1040 amino acid residues, isprovided. In a preferred aspect, a polypeptide which consists of 1038amino acid residues, is used.

In a further aspect, the polypeptides disclosed herein has a ratio oftransgalactosylation activity above 100% such as above 150%, 175% or200%.

Proteins are generally comprised of one or more functional regions,commonly termed domains. The presence of different domains in varyingcombinations in different proteins gives rise to the diverse repertoireof proteins found in nature. One way of describing the domains are bythe help of the Pfam database which is a large collection of proteindomain families as described in “The Pfam protein families database”: R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J. E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E. L.Sonnhammer, S. R. Eddy, A. Bateman Nucleic Acids Research (2010)Database Issue 38:D211-222. Each family is represented by multiplesequence alignments and hidden Markov models (HMMs). The herein providedpolypeptide(s) preferably contain one or more of the Pfam domainsGlyco_hydro2N (PF02837), Glyco_hydro (PF00703), Glyco_hydro 2C (PF02836)and Bacterial Ig-like domain (group 4) (PF07532). In one aspect, theherein provided polypeptide(s) contains Glyco_hydro2N (PF02837),Glyco_hydro (PF00703), Glyco_hydro 2C (PF02836) and Bacterial Ig-likedomain (group 4) (PF07532).

In one aspect, the polypeptides used herein have usefultransgalactosylating activity over a range of pH of 4-9, such as 5-8,such as 5.5-7.5, such as 6.5-7.5.

The present invention encompasses the use of polypeptides having acertain degree of sequence identity or sequence homology with amino acidsequence(s) defined herein or with a polypeptide having the specificproperties defined herein. The present invention encompasses, inparticular, the use of peptides having a degree of sequence identitywith any one of SEQ ID NO: 1, 2, 3, 4 or 5, defined below, or homologuesthereof.

The homologous amino acid sequence and/or nucleotide sequence shouldprovide and/or encode a polypeptide which retains the functionaltransgalactosylating activity and/or enhances the transgalactosylatingactivity compared to a polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 66%, 70%, 75%, 78%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99%, identical to the subject sequence.Typically, the homologues will comprise the same active sites etc. asthe subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

Thus, the present invention also encompasses the use of variants,homologues and derivatives of any amino acid sequence of a protein orpolypeptide as defined herein, particularly those of SEQ ID NO: 1, 2, 3,4 or 5 as defined below.

The sequences, particularly those of variants, homologues andderivatives of SEQ ID NO: 1, 2, 3, 4 or 5 defined below, may also havedeletions, insertions or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalentsubstance. Deliberate amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe secondary binding activity of the substance is retained. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

The present invention also encompasses conservative substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-conservativesubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Conservative substitutions that may be made are, for example within thegroups of basic amino acids (Arginine, Lysine and Histidine), acidicamino acids (glutamic acid and aspartic acid), aliphatic amino acids(Alanine, Valine, Leucine, Isoleucine), polar amino acids (Glutamine,Asparagine, Serine, Threonine), aromatic amino acids (Phenylalanine,Tryptophan and Tyrosine), hydroxyl amino acids (Serine, Threonine),large amino acids (Phenylalanine and Tryptophan) and small amino acids(Glycine, Alanine).

In one aspect, the polypeptide sequence used in the present invention isin a purified form.

In one aspect, the polypeptide or protein for use in the presentinvention is in an isolated form.

In one aspect, the polypeptide of the present invention is recombinantlyproduced.

The variant polypeptides include a polypeptide having a certain percent,e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, ofsequence identity with SEQ ID NO: 1 or 2.

The variant polypeptides include a polypeptide having a certain percent,e.g., at least 96%, 97%, 98%, or 99%, of sequence identity with SEQ IDNO: 3, 4 or 5.

In one aspect, the polypeptides employed herein comprises an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to the amino acid sequence of the maturepolypeptide encoded by the nucleotide sequence encoding thetransgalactosylase contained in Bifidobacterium bifidum DSM20215 shownherein as SEQ ID NO: 22. All considerations and limitations relating tosequence identities and functionality discussed in terms of the SEQ IDNO: 1, 2, 3, 4 or 5 apply mutatis mutandis to sequence identities andfunctionality of these polypeptides and nucleotides.

In one aspect, the subject amino acid sequence is SEQ ID NO: 1, 2, 3, 4or 5, and the subject nucleotide sequence preferably is SEQ ID NO: 9,10, 11, 12 or 13.

In one aspect, the polypeptide is a fragment having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofthe polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5; wherein the fragment hastransgalactosylating activity.

In one aspect, a fragment contains at least 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 amino acid residues.

In a further aspect, the length of the polypeptide variant is 500 to1300 amino acid residues. In a further aspect, the length of thepolypeptide variant is 600 to 1300 amino acids. In a further aspect, thelength of the polypeptide variant is 700 to 1300 amino acids. In afurther aspect, the length of the polypeptide variant is 800 to 1300amino acids. In a further aspect, the length of the polypeptide variantis 800 to 1300 amino acids.

Polypeptide Variants of SEQ ID NO: 1, 2, 3, 4 or 5

In one aspect, a variant of SEQ ID NO: 1, 2, 3, 4 or 5 having asubstitution at one or more positions which effects an altered propertysuch as improved transgalactosylation, relative to SEQ ID NO: 1, 2, 3, 4or 5, is used. Such variant polypeptides are also referred to in thisdocument for convenience as “variant polypeptide”, “polypeptide variant”or “variant”. In one aspect, the polypeptides as defined herein have animproved transgalactosylating activity as compared to the polypeptide ofSEQ ID NO: 1, 2, 3, 4 or 5. In another aspect, the polypeptides asdefined herein have an improved reaction velocity as compared to thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5.

The polypeptides and the variant polypeptides used herein comprisetransgalactosylation activity.

In one aspect, the ratio of transgalactosylating activity:β-galactosidase activity is at least 0.5, such as at least 1, such as atleast 1.5, or such as at least 2 after 30 min. reaction such as above aconcentration of 3% w/w initial lactose concentration.

In one aspect, the ratio of transgalactosylating activity:β-galactosidase activity is at least 2.5, such as at least 3, such as atleast 4, such as at least 5, such as at least 6, such as at least 7,such as at least 8, such as at least 9, such as at least 10, such as atleast 11, or such as at least 12 after 30 min. reaction such as above aconcentration of 3% w/w initial lactose concentration.

In one aspect, the polypeptides and the variants as defined herein arederivable from microbial sources, in particular from a filamentousfungus or yeast, or from a bacterium. The enzyme may, e.g., be derivedfrom a strain of Agaricus, e.g. A. bisporus; Ascovaginospora;Aspergillus, e.g. A. niger, A. awamori, A. foetidus, A. japonicus, A.oryzae; Candida; Chaetomium; Chaetotomastia; Dictyostelium, e.g. D.discoideum; Kluveromyces, e.g. K. fragilis, K. lactis; Mucor, e.g. M.javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa;Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus,R. stolonifer; Scierotinia, e.g. S. libertiana; Torula; Torulopsis;Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. scierotiorum;Bacillus, e.g. B. coagulans, B. circulans, B. megaterium, B. novalis, B.subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis;Bifidobacterium, e.g. B. longum, B. bifidum, B. animalis;Chryseobacterium; Citrobacter, e.g. C. freundii; Clostridium, e.g. C.perfringens; Diplodia, e.g. D. gossypina; Enterobacter, e.g. E.aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g. E.herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae;Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N. crassa; Proteus,e.g. P. vulgaris; Providencia, e.g. P. stuartii; Pycnoporus, e.g.Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, e.g. R.torques; Salmonella, e.g. S. typhimurium; Serratia, e.g. S.liquefasciens, S. marcescens; Shigella, e.g. S. flexneri; Streptomyces,e.g. S. antibioticus, S. castaneoglobisporus, S. violeceoruber;Trametes; Trichoderma, e.g. T. reesei, T. viride; Yersinia, e.g. Y.enterocolitica.

An isolated and/or purified polypeptide comprising a polypeptide or avariant polypeptide as defined herein is provided. In one embodiment,the variant polypeptide is a mature form of the polypeptide (SEQ ID NO:1, 2, 3, 4 or 5). In one aspect, the variants include a C-terminaldomain.

In one aspect, a variant polypeptide as defined herein includes variantswherein between one and about 25 amino acid residues have been added ordeleted with respect to SEQ ID NO: 1, 2, 3, 4 or 5. In one aspect, avariant polypeptide as defined herein includes variants wherein betweenone and 25 amino acid residues have been substituted, added or deletedwith respect to SEQ ID NO: 1, 2, 3, 4 or 5. In one aspect, the varianthas the amino acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5, wherein anynumber between one and about 25 amino acids have been substituted. In afurther aspect, the variant has the amino acid sequence of SEQ ID NO: 1,2, 3, 4 or 5, wherein any number between three and twelve amino acidshas been substituted. In a further aspect, the variant has the aminoacid sequence of SEQ ID NO: 1, 2, 3, 4 or 5, wherein any number betweenfive and nine amino acids has been substituted.

In one aspect, at least two, in another aspect at least three, and yetin another aspect at least five amino acids of SEQ ID NO: 1, 2, 3, 4 or5 have been substituted.

In one aspect, the herein disclosed polypeptide(s) has the sequence of1, 2, 3, 4 or 5.

In one aspect, the herein disclosed polypeptide(s) has the sequence ofSEQ ID NO: 1, 2, 3, 4 or 5, wherein the 10, such as 9, such as 8, suchas 7, such as 6, such 5, such as 4, such as 3, such as 2, such as 1amino acid in the N-terminal end are substituted and/or deleted.

Enzymes and enzyme variants thereof can be characterized by theirnucleic acid and primary polypeptide sequences, by three dimensionalstructural modelling, and/or by their specific activity. Additionalcharacteristics of the polypeptide or polypeptide variants as definedherein include stability, pH range, oxidation stability, andthermostability, for example. Levels of expression and enzyme activitycan be assessed using standard assays known to the artisan skilled inthis field. In another aspect, variants demonstrate improved performancecharacteristics relative to the polypeptide with SEQ ID NO: 1, 2, 3, 4or 5, such as improved stability at high temperatures, e.g., 65-85° C.

A polypeptide variant is provided as defined herein with an amino acidsequence having at least about 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5.

Nucleotides

In one aspect, the present invention employs isolated polypeptideshaving transgalactosylating activity as stated above which are encodedby polynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with i) the nucleic acid sequence comprised in SEQID NO: 9, 10, 11, 12 or 13 encoding the mature polypeptide of SEQ ID NO:1, 2, 3, 4 or 5; ii) the cDNA sequence of i) or iii) the complementarystrand of i) or ii), (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). A subsequence of SEQ ID NO: 9, 10, 11, 12 or 13 contains at least100 contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has lactase activity.

The nucleotide sequence of SEQ ID NO: 9, 10, 11, 12 or 13 or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 1,2, 3, 4 or 5 or a fragment thereof, may be used to design a nucleic acidprobe to identify and clone DNA encoding polypeptides havingtransgalactosylase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 14, preferably at least 25, more preferably at least 35, andmost preferably at least 70 nucleotides in length. It is, however,preferred that the nucleic acid probe is at least 100 nucleotides inlength. For example, the nucleic acid probe may be at least 200nucleotides, preferably at least 300 nucleotides, more preferably atleast 400 nucleotides, or most preferably at least 500 nucleotides inlength. Even longer probes may be used, e.g., nucleic acid probes whichare at least 600 nucleotides, at least preferably at least 700nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labelled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA library prepared from such other organisms may, therefore,be screened for DNA which hybridizes with the probes described above andwhich encodes a polypeptide having lactase activity. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 9,10, 11, 12 or 13 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labelled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 9, 10, 11,12 or 13, its complementary strand, or a subsequence thereof, under verylow to very high stringency conditions. Molecules to which the nucleicacid probe hybridizes under these conditions can be detected using X-rayfilm.

The nucleic acid probe may be the mature polypeptide coding region ofSEQ ID NO: 9, 10, 11, 12 or 13.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 g/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

In a particular embodiment, the wash is conducted using 0.2×SSC, 0.2%SDS preferably at least at 45° C. (very low stringency), more preferablyat least at 50° C. (low stringency), more preferably at least at 55° C.(medium stringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency). Inanother particular embodiment, the wash is conducted using 0.1×SSC, 0.2%SDS preferably at least at 45° C. (very low stringency), more preferablyat least at 50° C. (low stringency), more preferably at least at 55° C.(medium stringency), more preferably at least at 60° C. (medium-highstringency), even more preferably at least at 65° C. (high stringency),and most preferably at least at 70° C. (very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

Under salt-containing hybridization conditions, the effective T_(m) iswhat controls the degree of identity required between the probe and thefilter bound DNA for successful hybridization. The effective T_(m) maybe determined using the formula below to determine the degree ofidentity required for two DNAs to hybridize under various stringencyconditions.

Effective T _(m)=81.5+16.6(log M[Na⁺])+0.41(% G+C)−0.72(% formamide)

(See www.ndsu.nodak.edu/instruct/mcclean/pIsc731/dna/dna6.htm)

The G+C content of SEQ ID NO: 10 is 42% and the G+C content of SEQ IDNO: 11 is 44%. For medium stringency, the formamide is 35% and the Na⁺concentration for 5×SSPE is 0.75 M.

Another relevant relationship is that a 1% mismatch of two DNAs lowersthe T_(m) by 1.4° C. To determine the degree of identity required fortwo DNAs to hybridize under medium stringency conditions at 42° C., thefollowing formula is used:

% Homology=100−[(Effective T _(m)−Hybridization Temperature)/1.4]

(See www.ndsu.nodak.edu/instruct/mcclean/pIsc731/dna/dna6.htm)

The variant nucleic acids include a polynucleotide having a certainpercent, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, ofsequence identity with the nucleic acid encoding SEQ ID NO: 1, 2, 3, 4or 5. In one aspect, a nucleic acid capable of encoding a polypeptide asdisclosed herein, is provided. In a further aspect, the herein disclosednucleic acid has a nucleic acid sequence which is at least 60%, such asat least 65%, such as at least 70%, such as at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as at least 99% identical SEQ ID NO: 9, 10, 11, 12 or 13.

In one aspect, a plasmid comprising a nucleic acid as described hereinmay be used. In another aspect, an expression vector comprising anucleic acid as described herein, or capable of expressing a polypeptideas described herein may be used.

A nucleic acid complementary to a nucleic acid encoding any of thepolypeptide variants as defined herein set forth herein is provided.Additionally, a nucleic acid capable of hybridizing to the complement isprovided. In another embodiment, the sequence for use in the methods andcompositions described here is a synthetic sequence. It includes, but isnot limited to, sequences made with optimal codon usage for expressionin host organisms, such as yeast. The polypeptide variants as providedherein may be produced synthetically or through recombinant expressionin a host cell, according to procedures well known in the art. In oneaspect, the herein disclosed polypeptide(s) is recombinantpolypeptide(s). The expressed polypeptide variant as defined hereinoptionally is isolated prior to use.

In another embodiment, the polypeptide variant as defined herein ispurified following expression. Methods of genetic modification andrecombinant production of polypeptide variants are described, forexample, in U.S. Pat. Nos. 7,371,552, 7,166,453; 6,890,572; and6,667,065; and U.S. Published Application Nos. 2007/0141693;2007/0072270; 2007/0020731; 2007/0020727; 2006/0073583; 2006/0019347;2006/0018997; 2006/0008890; 2006/0008888; and 2005/0137111. The relevantteachings of these disclosures, including polypeptide-encodingpolynucleotide sequences, primers, vectors, selection methods, hostcells, purification and reconstitution of expressed polypeptidevariants, and characterization of polypeptide variants as definedherein, including useful buffers, pH ranges, Ca²⁺ concentrations,substrate concentrations and enzyme concentrations for enzymatic assays,are herein incorporated by reference.

A nucleic acid sequence is provided encoding the protein of SEQ ID NO:1, 2, 3, 4 or 5 or a nucleic acid sequence having at least about 66%,68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with a nucleic acid encoding the protein of SEQ ID NO:1, 2, 3, 4 or 5. In one embodiment, the nucleic acid sequence has atleast about 60%, 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the nucleic acid of SEQ ID NO: 9,10, 11, 12 or 13.

Vectors

In one aspect, the invention employs a vector comprising apolynucleotide. In one aspect, a bacterial cell comprises the vector. Insome embodiments, a DNA construct comprising a nucleic acid encoding avariant is transferred to a host cell in an expression vector thatcomprises regulatory sequences operably linked to an encoding sequence.The vector may be any vector that can be integrated into a fungal hostcell genome and replicated when introduced into the host cell. The FGSCCatalogue of Strains, University of Missouri, lists suitable vectors.Additional examples of suitable expression and/or integration vectorsare provided in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (2001); Bennett et al., MORE GENE MANIPULATIONS IN FUNGI, AcademicPress, San Diego (1991), pp. 396-428; and U.S. Pat. No. 5,874,276.Exemplary vectors include pFB6, pBR322, PUC18, pUC100 and pENTR/D,pDON™201, pDONR™221, pENTR™, pGEM®3Z and pGEM®4Z. Exemplary for use inbacterial cells include pBR322 and pUC19, which permit replication in E.coli, and pE194, for example, which permits replication in Bacillus.

In some embodiments, a nucleic acid encoding a variant is operablylinked to a suitable promoter, which allows transcription in the hostcell. The promoter may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, and egl2 promoters. Inone embodiment, the promoter is one that is native to the host cell. Forexample, when P. saccharophila is the host, the promoter is a native P.saccharophila promoter. An “inducible promoter” is a promoter that isactive under environmental or developmental regulation. In anotherembodiment, the promoter is one that is heterologous to the host cell.

In some embodiments, the coding sequence is operably linked to a DNAsequence encoding a signal sequence. In another aspect, a representativesignal peptide is SEQ ID NO: 27. A representative signal peptide is SEQID NO: 9 which is the native signal sequence of the Bacillus subtilisaprE precursor. In other embodiments, the DNA encoding the signalsequence is replaced with a nucleotide sequence encoding a signalsequence from other extra-cellular Bacillus subtilis pre-cursors. In oneembodiment, the polynucleotide that encodes the signal sequence isimmediately upstream and in-frame of the polynucleotide that encodes thepolypeptide. The signal sequence may be selected from the same speciesas the host cell. In additional embodiments, a signal sequence and apromoter sequence comprising a DNA construct or vector to be introducedinto a fungal host cell are derived from the same source. In someembodiments, the expression vector also includes a termination sequence.In one embodiment, the termination sequence and the promoter sequenceare derived from the same source. In another embodiment, the terminationsequence is homologous to the host cell.

In some embodiments, an expression vector includes a selectable marker.Examples of suitable selectable markers include those that conferresistance to antimicrobial agents, e.g., hygromycin or phleomycin.Nutritional selective markers also are suitable and include amdS, argB,and pyr4. In one embodiment, the selective marker is the amdS gene,which encodes the enzyme acetamidase; it allows transformed cells togrow on acetamide as a nitrogen source. The use of an A. nidulans amdSgene as a selective marker is described in Kelley et al., EMBO J. 4:475-479 (1985) and Penttila et al., Gene 61: 155-164 (1987).

A suitable expression vector comprising a DNA construct with apolynucleotide encoding a variant may be any vector that is capable ofreplicating autonomously in a given host organism or integrating intothe DNA of the host. In some embodiments, the expression vector is aplasmid. In some embodiments, two types of expression vectors forobtaining expression of genes are contemplated. The first expressionvector comprises DNA sequences in which the promoter, coding region, andterminator all originate from the gene to be expressed. In someembodiments, gene truncation is obtained by deleting undesired DNAsequences to leave the domain to be expressed under control of its owntranscriptional and translational regulatory sequences. The second typeof expression vector is preassembled and contains sequences required forhigh-level transcription and a selectable marker. In some embodiments,the coding region for a gene or part thereof is inserted into thisgeneral-purpose expression vector, such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

Expression Hosts/Host Cells

In a further aspect, a host cell comprising, preferably transformedwith, a plasmid as described herein or an expression vector as describedherein, is used.

In a further aspect, a cell capable of expressing a polypeptide asdescribed herein, is used.

In one aspect, the host cell as described herein, or the cell asdescribed herein is a bacterial, fungal or yeast cell.

In a further aspect, the host cell is selected from the group consistingof Ruminococcus, Bifidobacterium, Lactococcus, Lactobacillus,Streptococcus, Leuconostoc, Escherichia, Bacillus, Streptomyces,Saccharomyces, Kluyveromyces, Candida, Torula, Torulopsis andAspergillus.

In a further aspect, the host cell is selected from the group consistingof Ruminococcus hansenii, Bifidobacterium breve, Bifidobacterium longum,Bifidobacterium infantis, Bifidobacterium bifidum and Lactococcuslactis.

In another embodiment, suitable host cells include a Gram positivebacterium selected from the group consisting of Bacillus subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. thuringiensis, Streptomyces lividans, or S. murinus; or aGram negative bacterium, wherein said Gram negative bacterium isEscherichia coli or a Pseudomonas species. In one aspect, the host cellis a B. subtilus or B. licheniformis. In one embodiment, the host cellis B. subtilis, and the expressed protein is engineered to comprise a B.subtilis signal sequence, as set forth in further detail below. In oneaspect, the host cell expresses the polynucleotide as set out in theclaims.

In some embodiments, a host cell is genetically engineered to express apolypeptide variant as defined herein with an amino acid sequence havingat least about 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the polypeptide ofSEQ ID NO: 1, 2, 3, 4 or 5. In some embodiments, the polynucleotideencoding a polypeptide variant as defined herein will have a nucleicacid sequence encoding the protein of SEQ ID NO: 1, 2, 3, 4 or 5 or anucleic acid sequence having at least about 66%, 68%, 70%, 72%, 74%,78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identitywith a nucleic acid encoding the protein of SEQ ID NO: 1, 2, 3, 4 or 5.In one embodiment, the nucleic acid sequence has at least about 60%,66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity to the nucleic acid of SEQ ID NO: 9, 10, 11, 12 or 13.

Methods for Producing Polypeptides

In a further aspect, a method of expressing a polypeptide as describedherein comprises obtaining a host cell or a cell as described herein andexpressing the polypeptide from the cell or host cell, and optionallypurifying the polypeptide. Such a polypeptide may be used in the presentinvention.

An expression characteristic means an altered level of expression of thevariant, when the variant is produced in a particular host cell.Expression generally relates to the amount of active variant that isrecoverable from a fermentation broth using standard techniques known inthis art over a given amount of time. Expression also can relate to theamount or rate of variant produced within the host cell or secreted bythe host cell. Expression also can relate to the rate of translation ofthe mRNA encoding the variant polypeptide.

Transformation, Expression and Culture of Host Cells

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, e.g., lipofection mediatedand DEAE-Dextrin mediated transfection; incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are known in the art. See, e.g., Ausubel et al. (1987),supra, chapter 9; Sambrook et al. (2001), supra; and Campbell et al.,Curr. Genet. 16: 53-56 (1989). The expression of heterologous protein inTrichoderma is described, for example, in U.S. Pat. Nos. 6,022,725;6,268,328; Harkki et al., Enzyme Microb. Technol. 13: 227-233 (1991);Harkki et al., BioTechnol. 7: 596-603 (1989); EP 244,234; and EP215,594. In one embodiment, genetically stable transformants areconstructed with vector systems whereby the nucleic acid encoding avariant is stably integrated into a host cell chromosome. Transformantsare then purified by known techniques.

In one non-limiting example, stable transformants including an amdSmarker are distinguished from unstable transformants by their fastergrowth rate and the formation of circular colonies with a smooth, ratherthan ragged outline on solid culture medium containing acetamide.Additionally, in some cases a further test of stability is conducted bygrowing the transformants on solid non-selective medium, e.g., a mediumthat lacks acetamide, harvesting spores from this culture medium anddetermining the percentage of these spores that subsequently germinateand grow on selective medium containing acetamide. Other methods knownin the art may be used to select transformants.

Identification of Activity

To evaluate the expression of a variant in a host cell, assays canmeasure the expressed protein, corresponding mRNA, or β-galactosidaseactivity. For example, suitable assays include Northern and Southernblotting, RT-PCR (reverse transcriptase polymerase chain reaction), andin situ hybridization, using an appropriately labeled hybridizing probe.Suitable assays also include measuring activity in a sample. Suitableassays of the activity of the variant include, but are not limited to,ONPG based assays or determining glucose in reaction mixtures such forexample described in the methods and examples herein.

Methods for Purifying Herein Disclosed Polypeptides

In general, a variant produced in cell culture is secreted into themedium and may be purified or isolated, e.g., by removing unwantedcomponents from the cell culture medium. In some cases, a variant may berecovered from a cell lysate. In such cases, the enzyme is purified fromthe cells in which it was produced using techniques routinely employedby those of skill in the art. Examples include, but are not limited to,affinity chromatography, ion-exchange chromatographic methods, includinghigh resolution ion-exchange, hydrophobic interaction chromatography,two-phase partitioning, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin, such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using Sephadex G-75, for example. The herein disclosedpolypeptide(s) is spray-dried.

Spray Drying

In general terms, spray drying is a process for producing powders from aliquid where a suspension or solution is fed to an atomizer and thedroplets formed are mixed with a hot gas. Generally the polypeptide ofthe invention will be in solution. The solvent of the droplets thusevaporate, leaving dry particles. Conventional spray-drying techniquesmay be used in the processing of the present invention, such as thosediscussed in Spray-Drying Handbook, 4^(th) Edition, K. Masters, (1985),which is incorporated herein by reference. A simple spray drier plantdesign has three process stages which characterise spray drying:

1) Feed atomization;

2) Droplet drying by mixing of drying gas and spray; and

3) Separation of drying gas and spray.

The method for the preparation of a spray-dried powder typically firstcomprises the dispersion of a carrier in water, and then the mixture ofthis dispersion. The mixture is then spray-dried to produce a powderedproduct.

Thus, particle drying according to the present invention is performedthrough a spray-drying process. In its most basic form, the processinvolves the following: transporting a liquid or suspension through anatomizing device into a drying chamber; mixing droplets of the atomizedliquid or suspension with a stream of heated air; evaporating volatilecomponents of the droplets in the stream of air leaving dried particles.

The atomizer may be of any suitable type. Non-limiting examples ofatomizers include high speed rotating disk atomizers, pressure nozzleatomizers, pneumatic nozzle atomizers, and sonic nozzle atomizers.

The spray-dried powder of the invention can also be advantageously usedas an intermediate product or starting product for adouble-encapsulation method, i.e. as a solid product susceptible ofbeing subjected to a further encapsulation such as an extrusion in aglassy matrix to provide a granular delivery system, or to a secondspray drying operation in a distinct or similar matrix, and theinvention also relates to that use of the composition.

The spray-drying apparatus used in the process of the invention can beany one of the various commercially available apparatuses. Examples ofspray-drying apparatuses are the Anhydro Dryers (origin: Anhydro Corp.of Attleboro Falls, Mass.), the Niro Dryer (manufactured by NiroAtomizer Ltd., Copenhagen, Denmark), or a Leaflash apparatus (origin:CCM Sulzer). Preferably a spray-drier with a pressure nozzle is used.

The typical parameters of a spray-drying process are well known in theart and can be easily adjusted by a skilled person in the art.

The particles of the invention have typically a size comprised between50 and 70 μm and a bulk density comprised between 0.4 and 0.6 g/cm3.

However, the granulometry and the bulk density of the resulting drypowders can be adjusted by inter alia selecting the nozzle (orificesize/diameter) and the atomization pressure so as to obtain the desiredpowder flowability.

The compositions of the invention may also contain optional ingredientsin addition to the maltodextrin and/or sodium chloride.

The composition of the fluid going into the spray dryer may beformulated so that the atomized liquid, generally aqueous, compositionformed in the initial stages of the spray-drying process includes atleast one polypeptide, which is typically present in the liquidcomposition at a concentration greater than 0.01 or greater than 0.5weight percent.

In a method aspect, the present invention provides a method forincreasing the yield of a spray-drying process. The process provides aparticle that includes a polypeptide at a concentration typicallygreater than 0.5 weight percent. The method includes the followingsteps: a) feeding an aqueous composition into a spray-drying apparatus,wherein the aqueous composition comprises a maltodextrin and/or sodiumchloride and at least one polypeptide and, wherein the at least onepolypeptide is present in the aqueous composition at a concentrationtypically of 20 to 80 g/L; and, b) spray-drying the composition. In theaqueous composition, the sodium chloride should preferably be present ata level so as to give microbial stability to the final spray-driedcomposition, therefore a range of 10-20% in the aqueous compositionwould be suitable, with a maximum of about 25%. The maltodextrin may beused in the aqueous composition in the range of 5-40%, with 10-30% beingpreferred. In one embodiment the maltodextrin is used at about 17%.

An enzyme-containing liquid or suspension may be used in the presentinvention and may be, for example, a fermentation broth or processedfermentation broth.

A fermentation broth includes microbial cells and/or related cell debris(i.e., biomass). Some or most of the biomass may be removed from thefermentation broth to modify properties of the broth for spray drying.Typically, at least 10 percent by weight to 20 percent by weight of thebiomass is removed from the broth prior to spray drying. Oftentimes, atleast 30 percent, 40 percent, 50 percent, or 60 percent of the biomassis removed, and in certain cases at least 70 percent, 80 percent, 90percent, or 95 percent of the biomass is removed.

Biomass may be removed from the fermentation broth using a variety oftechniques. Such techniques include filtration, centrifugation,flocculation and combinations thereof. Typically, the fermentation brothincludes between 0 and 35 percent weight/weight dry matter. Oftentimes,the broth includes between 0 and 20 percent weight/weight dry matter orbetween 0 and 15 percent weight/weight dry matter. In certain cases, thefermentation broth includes between 5 percent and 15 percentweight/weight dry matter. Up to 90 percent weight/weight of the drymatter is biomass. Oftentimes, up to 75 percent, 50 percent or 25percent weight/weight of the dry matter is biomass. In certain cases, upto 10 percent weight/25 weight of the dry matter is biomass.

The fermentation broth may be de-sludged through the removal of coarseparticles or bodies. Such particles/bodies include straw, rubble, soygrits and other non-biomass insolubles that typically originate fromnutrients added to the broth during fermentation. Removal is typicallyaccomplished by one of the following methods: straining, filtration,sedimentation, centrifugation and/or decanting the broth.

Where a solution or suspension containing an enzyme is used in thepresent invention, the liquid medium is typically water. For instance,the enzyme-containing material may be enzyme concentrate obtained fromfermentation filtrate processing. Processing methods used to concentratethe fermentation broth include, without limitation: ultra filtration toreduce water content and low molecular components; extraction of theenzyme from the fermentation filtrate into a second liquid;crystallization or precipitation of the enzyme followed by resuspensionand, purification through column chromatography may be used, e.g. bypumping 5 the fermentation filtrate through a column comprising a resin.

Materials may be added to an enzyme-containing liquid to improve theproperties of spray dried products obtained from the liquids.Non-limiting examples of such additives include: salts (e.g., alkalisalts, earth metal salts, additional chloride salts, sulfate salts,nitrate salts, carbonate salts, where exemplary counterions are calcium,potassium, and sodium), inorganic minerals or clays (e.g., zeolites,kaolin, bentonite, talc's and/or silicates), carbohydrates (e.g.,sucrose and/or starch), coloring pigments (e.g., titanium dioxide),biocides (e.g., Rodalon®, Proxel®), dispersants, anti-foaming agents,acid agents, alkaline agents, enzyme stabilizers (e.g., methionine, orthiosulphate), enzyme inhibitors (e.g., boric acid protease inhibitors),binders other enzymes and combinations thereof. Polymeric additivestypically are either low MW 15 (<250,000 Daltons) materials, or areadded as slurries where the additive is not in solution.

It is preferable that a process is provided in which the dust levelsproduced are at a minimum. We have found that advantageously a reductionin dust levels can be achieved through the use of potato starch in theprocess. Thus in one embodiment a potato starch is added to theenzyme-containing liquid to improve the size distribution of spray driedproducts obtained from the liquids. In such an embodiment, thecomposition prepared according to the spray-dried process may comprise5-50% by weight of maltodextrin or sodium chloride, 25-95% of the potatostarch and 20-45% by weight of the enzyme wherein in any one formulationthe total amount of components (which may include additional componentsto those recited above) equals 100% by weight.

The enzyme-containing liquid may also be subjected to physicaltreatments prior to spray drying. Such physical treatments include,without limitation, heating and/or cooling and/or radiating the liquid,mixing the liquid, aerating the liquid, and ultra-sound treatment of theliquid. Enzyme-containing liquids used in the present inventiontypically include at least 1 mg of “active” enzyme, e.g. catalyticallyactive protein of interest, per liter of liquid. Typically, the liquidsinclude about 20 g/L to 80 g/L active polypeptide, which corresponds toabout 500 to 2000 LAU/g active polypeptide.

Post Processing of Spray-Dried Particles

The spray-dried particles formed according to the present invention maybe further processed using a variety of methods. Non-limiting examplesof such methods include mixer granulation, prilling, extrusion, fluidbed processes, coating, and milling/grinding and screening. Mixergranulation involves mixing spray dried particles with water and anadditional component. Additional components are typically binders,fibers, salts, water insoluble minerals, pigments, enzyme stabilizers orcombinations thereof. Water is added in amounts sufficient toagglomerate solid components into granules of a suitable mean size. Thewater is subsequently removed using a suitable drying method. Bindersused in a mixer granulation process for particles of the presentinvention are polymeric in nature. Exemplary binders include polyvinylpyrrolidone, dextrins and cellulose derivatives (e.g., hydroxypropylcellulose, methyl cellulose or carboxymethyl cellulose Glucidex 21D,available from Roquette Freres, France, is oftentimes a suitable binder.Fibers used in a mixer granulation process include pure and/or impurefibrous cellulose, such as sawdust, pure fibrous cellulose, and cotton.Filter aids based on fibrous cellulose can also be used. Examples ofcommercially available fibrous cellulose include Cepo™ and Arbocell™.Synthetic fibers as discussed in EP 304331 B1 may be used, includingfibers made of polyethylene, polypropylene, polyester, especially nylon,polyvinylformate, poly(meth)acrylic compounds. Salts used in a mixergranulation process include water soluble and/or insoluble salts such asalkali and/or earth alkali salts of sulfate, chloride, carbonate andphosphate.

Water insoluble minerals used in a mixer granulation process includezeolites, clays like kaolin and bentonite, tales, and/or silicates.Pigments used in a mixer granulation process include titanium dioxide.

Enzyme stabilizers used in a mixer granulation process include alkalineor neutral materials (e.g., metal silicates, carbonates orbicarbonates), reducing agents (e.g., sulfite, thiosulfite, orthiosulfate), antioxidants (e.g., methionine, butylated hydroxytoluene,or butylated hydroxyanisol) and/or salts of first transition seriesmetal ions. These agents may be used in conjunction with otherprotective agents of the same or different categories. A number of mixergranulation process are known in the art, including those discussed inthe following documents: U.S. Pat. No. 4,106,991; EP 170360 B1; EP304332 B1; EP 304331; WO 90/09440; and, WO 90/09428.

Prilling involves suspending dried particles in molten wax followed byspray cooling of the suspension. The process is discussed in Michael S.Showell (editor); Powdered detergents; Surfactant Science Series; 1998;vol. 71, page 140-142, Marcel Dekker; and, DK-PA 1999. A wax used in theprilling process has a melting point between 25 and 125° C. and istypically an organic compound or a salt of an organic compound. Itoftentimes is either water soluble or water dispersible in a neutral oralkaline solution. Non-limiting examples of water soluble waxes are thepolyethylene glycols (e.g., PEG 1000).

Extrusion involves adding moisture to particles, either alone or mixedwith an additive as described for mixer granulation, to provide a paste.The paste is pressed into pellets or is extruded under pressure througha small opening; it is then cut into particles, which are dried.Extrusion processes are discussed in Michael S. Showell (editor);Powdered detergents; Surfactant Science Series; 1998; vol. 71, page140-42, Marcel Dekker; and, U.S. Pat. No. 4,661,452.

Fluid bed processes involve fluidizing spray dried particles in a fluidbed. A solution containing a binder is atomized and brought into contactwith the fluidized particles. This causes the particles to bindtogether, forming larger, stronger particles. Spray dried particles ofthe present invention may be coated with one or more coating layers. Thecoating may include materials such as binders, fibers, salts, waterinsoluble materials, pigments, enzyme stabilizers or combinationsthereof as described above in the mixer granulation section.

The processes described above may be supplemented with milling/grindingand/or screening processes at any stage. It may, for example, bedesirable to grind the spray dried particles prior to subsequentprocessing steps and to screen the final product to obtain the desiredsize fraction.

Compositions, Application and Use

Examples are given below of preferred uses of the polypeptides orpolypeptide-containing compositions of the invention.

In one aspect, disclosed herein is a method for producing a food productby treating a substrate comprising lactose with a spray-driedcomposition as described herein.

In one aspect, disclosed herein is a method for producing a dairyproduct by treating a milk-based substrate comprising lactose with aspray-dried composition as described herein.

In one aspect, the substrate comprising lactose is further treated witha hydrolysing beta-galactosidase.

In one aspect, a composition preferably a food composition, morepreferably a dairy product comprising a cell or a polypeptide asdescribed herein, is provided.

Furthermore, disclosed herein is a composition comprising at least 5%,such as e.g. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% w/w of one ormore polypeptide(s) as disclosed herein based on the total amount ofpolypeptides in the composition having at least 70%, e.g. such as 72%,74%, 74%, 78%, 80%, 82%, 84%, 86%, 88%, 90% sequence identity with SEQID NO: 22. This may be evaluated by using the following techniques knowto a person skilled in the art. The samples to be evaluated aresubjected to SDS-PAGE and visualized using a dye appropriate for proteinquantification, such as for example the Bio-Rad Criterion system. Thegel is then scanned using appropriate densiometic scanner such as forexample the Bio-Rad Criterion system and the resulting picture isensured to be in the dynamic range. The bands corresponding to anyvariant/fragment derived from SEQ ID NO: 8 are quantified and thepercentage of the polypeptides are calculated as: Percentage ofpolypeptide in question=polypeptide in question/(sum of all polypeptidesexhibiting transgalactosylating activity)*100. The total number ofpolypeptides variants/fragments derived from SEQ ID NO:8 in thecomposition can be determined by detecting fragment derived from SEQ IDNO:8 by western blotting using a polyclonal antibody by methods know toa person skilled in the art.

In one aspect, the composition according to the present inventioncomprises one or more polypeptide(s) selected from the group consistingof a polypeptide consisting of SEQ ID NO: 1, 2, 3, 4 and 5. In a furtheraspect, the composition comprises one or more polypeptide(s) selectedfrom the group consisting of a polypeptide consisting of SEQ ID NO: 1, 2and 3. In yet a further aspect, the composition comprises one or morepolypeptide(s) selected from the group consisting of a polypeptideconsisting of SEQ ID NO: 1 and 2.

In one aspect the invention provides an enzyme complex preparationcomprising the enzyme complex according to the invention, an enzymecarrier in the form of maltodextrin and/or sodium chloride andoptionally a stabilizer and/or a preservative.

In yet a further aspect of the invention, the composition does notinclude a polyol, such as glycerol, or water.

In a further aspect, the preparation/composition comprises a stabilizer.In one aspect, the stabilizer is selected from the group consisting ofinorganic salts, sugars and combinations thereof. In one aspect, thestabilizer is an inorganic salt such as potassium chloride. In anotheraspect, the stabilizer is not a polyol such as glycerol, propyleneglycol, or sorbitol. In yet another aspect, the sugar is asmall-molecule carbohydrate, in particular any of several sweet-tastingones such as glucose, galactose, fructose and saccharose.

In yet at further aspect, the preparation comprises a preservative. Inone aspect, the preservative is methyl paraben, propyl paraben,benzoate, sorbate or other food approved preservatives or a mixturethereof.

Excipients which may be used in the preparation/composition includemaltose, sucrose, glucose including glucose syrup or dried glucosesyrup, pre-cooked starch, gelatinised starch, L-lactic, ascorbylpalmitate, tocopherols, lecithins, citric acid, citrates, phosphoric,phosphates, sodium alginate, carrageenan, locust bean gum, guar gum,xanthan gum, pectins, sodium carboxymethylcellulose, mono- anddiglycerides, citric acid esters of mono- and diglycerides, sucroseesters, carbon dioxide, argon, helium, nitrogen, nitrous oxide, oxygen,hydrogen, and starch sodium octenylsuccinate.

In one aspect, a method for producing a dairy product by treating amilk-based substrate comprising lactose with a spray-dried compositionas described herein is provided. In a further aspect, a method forproducing a dairy product by treating a milk-based substrate comprisinglactose with a polypeptide having a relative transgalactosylationactivity above 60%, such as above 70%, such as above 75% after 15 min.reaction, is provided. In one aspect, the relative transgalactosylationactivity is above 3 after 30 min. reaction. In a further aspect, therelative transgalactosylation activity is above 6 after 30 min.reaction. In yet a further aspect, the relative transgalactosylationactivity is above 12 after 30 min. reaction. In one aspect, a method isprovided, wherein the treatment with a polypeptide as described hereintakes place at an optimal temperature for the activity of the enzyme. Ina further aspect, the polypeptide is added to the milk-based substrateat a concentration of 0.01-1000 ppm. In yet a further aspect, thepolypeptide is added to the milk-based substrate at a concentration of0.1-100 ppm. In a further aspect, the polypeptide is added to themilk-based substrate at a concentration of 1-10 ppm. In one aspect, amethod further comprising fermenting a substrate such as a dairy productwith a microorganism, is provided. In a further aspect, the dairyproduct is yogurt. In a further aspect, the treatment with thepolypeptide and the microorganism is performed essentially at the sametime. In one aspect, the polypeptide and the microorganism are added tothe milk-based substrate essentially at the same time.

In one aspect, a dairy product comprising a spray-dried composition asdescribed herein, is provided. In one aspect, the polypeptide as definedherein is added in a concentration of 0.01-1000 ppm. In one aspect, adairy product comprising an inactivated polypeptide as defined herein,is provided. In one aspect, a dairy product comprising GOS formed insitu by a polypeptide as defined herein, is provided. In one aspect, adairy product comprising a cell as defined herein, is provided.

A dairy product as described herein may be, e.g., skim milk, low fatmilk, whole milk, cream, UHT milk, milk having an extended shelf life, afermented milk product, cheese, yoghurt, butter, dairy spread, buttermilk, acidified milk drink, sour cream, whey based drink, ice cream,condensed milk, dulce de leche or a flavoured milk drink. A dairyproduct may be manufactured by any method known in the art.

A dairy product may additionally comprise non-milk components, e.g.vegetable components such as, e.g., vegetable oil, vegetable protein,and/or vegetable carbohydrates. Dairy products may also comprise furtheradditives such as, e.g., enzymes, flavouring agents, microbial culturessuch as probiotic cultures, salts, sweeteners, sugars, acids, fruit,fruit juices, or any other component known in the art as a component of,or additive to, a dairy product.

In one embodiment of the invention, one or more milk components and/ormilk fractions account for at least 50% (weight/weight), such as atleast 70%, e.g. at least 80%, preferably at least 90%, of the dairyproduct.

In one embodiment of the invention, one or more milk-based substrateshaving been treated with an enzyme as defined herein havingtransgalactosylating activity account for at least 50% (weight/weight),such as at least 70%, e.g. at least 80%, preferably at least 90%, of thedairy product.

In one embodiment of the invention, the dairy product is a dairy productwhich is not enriched by addition of pre-producedgalacto-oligosaccharides.

In one embodiment of the invention, the polypeptide-treated milk-basedsubstrate is not dried before being used as an ingredient in the dairyproduct.

In one embodiment of the invention, the dairy product is ice cream. Inthe present context, ice cream may be any kind of ice cream such as fullfat ice cream, low fat ice cream, or ice cream based on yoghurt or otherfermented milk products. Ice cream may be manufactured by any methodknown in the art.

In one embodiment of the invention, the dairy product is milk orcondensed milk.

In one embodiment of the invention, the dairy product is UHT milk. UHTmilk in the context of the present invention is milk which has beensubjected to a sterilization procedure which is intended to kill allmicroorganisms, including the bacterial spores. UHT (ultra hightemperature) treatment may be, e.g., heat treatment for 30 seconds at130° C., or heat treatment for one second at 145° C.

In one preferred embodiment of the invention, the dairy product is ESLmilk. ESL milk in the present context is milk which has an extendedshelf life due to microfiltration and/or heat treatment and which isable to stay fresh for at least 15 days, preferably for at least 20days, on the store shelf at 2-5° C.

In another preferred embodiment of the invention, the dairy product is afermented dairy product, e.g., yoghurt.

The microorganisms used for most fermented milk products are selectedfrom the group of bacteria generally referred to as lactic acidbacteria. As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of anaerobic bacteria, bifidobacteria, i.e.Bifidobacterium spp., which are frequently used as food cultures aloneor in combination with lactic acid bacteria, are generally included inthe group of lactic acid bacteria.

Lactic acid bacteria are normally supplied to the dairy industry eitheras frozen or freeze-dried cultures for bulk starter propagation or asso-called “Direct Vat Set” (DVS) cultures, intended for directinoculation into a fermentation vessel or vat for the production of afermented dairy product. Such cultures are in general referred to as“starter cultures” or “starters”.

Commonly used starter culture strains of lactic acid bacteria aregenerally divided into mesophilic organisms having optimum growthtemperatures at about 30° C. and thermophilic organisms having optimumgrowth temperatures in the range of about 40 to about 45° C. Typicalorganisms belonging to the mesophilic group include Lactococcus lactis,Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp.cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcuspentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis,Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp.paracasei. Thermophilic lactic acid bacterial species include asexamples Streptococcus thermophilus, Enterococcus faecium, Lactobacillusdelbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillusdelbrueckii subsp. bulgaricus and Lactobacillus acidophilus. Also theanaerobic bacteria belonging to the genus Bifidobacterium includingBifidobacterium bifidum, Bifidobacterium animalis and Bifidobacteriumlongum are commonly used as dairy starter cultures and are generallyincluded in the group of lactic acid bacteria. Additionally, species ofPropionibacteria are used as dairy starter cultures, in particular inthe manufacture of cheese. Additionally, organisms belonging to theBrevibacterium genus are commonly used as food starter cultures.

Another group of microbial starter cultures are fungal cultures,including yeast cultures and cultures of filamentous fungi, which areparticularly used in the manufacture of certain types of cheese andbeverage. Examples of fungi include Penicillium roqueforti, Penicilliumcandidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir andSaccharomyces cerevisiae.

In one embodiment of the present invention, the microorganism used forfermentation of the milk-based substrate is Lactobacillus casei or amixture of Streptococcus thermophilus and Lactobacillus delbrueckiisubsp. bulgaricus.

Fermentation processes to be used in a method of the present inventionare well known and the person of skill in the art will know how toselect suitable process conditions, such as temperature, oxygen, amountand characteristics of microorganism/s, additives such as e.g.carbohydrates, flavours, minerals, enzymes, and process time. Obviously,fermentation conditions are selected so as to support the achievement ofthe present invention.

As a result of fermentation, pH of the milk-based substrate will belowered. The pH of a fermented dairy product of the invention may be,e.g., in the range 3.5-6, such as in the range 3.5-5, preferably in therange 3.8-4.8.

In one aspect, a method of using the spray-dried composition is providedto produce galacto-oligosaccharides,

In one embodiment of the invention, the GOS is produced by incubatingthe spray-dried composition in a medium that comprises a lactosesubstrate. The incubation is carried out under conditions where GOS isproduced.

In one aspect, the use of a herein disclosed spray-dried composition forproducing a product selected from the group consisting of yoghurt,cheese, fermented milk product, dietary supplement and probioticcomestible product, is provided.

In one aspect, the spray-dried composition described herein may be usedto prepare cheese products and in methods for making the cheeseproducts. Cheese products may e.g. be selected from the group consistingof cream cheese, cottage cheese, and process cheese. By addingpolypeptides the cheeses may contain significantly increased levels ofgalacto-oligosaccharides and reduced levels of lactose. In one aspect,the lactose levels in the final cheese product may be reduced by atleast about 25 percent, preferably at least about 50 percent, and morepreferably at least about 75 percent. The polypeptides may be used toreduce lactose in cheese products to less than about 1 gram per serving,an amount that can be tolerated by most lactose-intolerant individuals.

The cheese products provided herein are nutritionally-enhanced cheeseproducts having increased soluble fiber content, reduced caloriccontent, excellent organoleptic properties, improved texture, andflavor. Further, the polypeptides described herein may reduce theglycemic index of the cheese products because GOS are more slowlyabsorbed than lactose or its hydrolysis products. Finally, thepolypeptides may reduce the cost of production of cheese products,particularly cream cheese products, because GOS surprisingly provideimproved texture to the cream cheese product, thus permitting reduceduse of stabilizers, or by allowing for increased moisture contentwithout syneresis.

In a further aspect, a composition comprising a spray-dried compositionas described herein and a carbohydrate substrate, is provided. In afurther aspect, the carbohydrate substrate is a disaccharide. In afurther aspect, the disaccharide is for example lactulose, trehalose,rhamnose, maltose, sucrose, lactose or cellobiose. In yet a furtheraspect, the carbohydrate substrate is lactose. The composition isprepared such that oligosaccarides are produced. The polypeptide asdescribed herein may be part of a product selected from the groupconsisting of yoghurt, cheese, fermented milk products, dietarysupplements, and probiotic comestible products. In one aspect, acomposition comprising a polypeptide as described herein and astabilizer, is provided. Examples of stabilizers are a sugar or a sugaralcohol, lactic acid, boric acid, or a boric acid derivative (e.g., anaromatic borate ester). Preferably the stabilizer is not a polyol suchas, e.g. glycerol or propylene glycol.

In one aspect, the use of a transgalactosylating polypeptide in acomposition as disclosed herein, for producing galacto-oligosaccharides,is provided. In one aspect, the use of a spray-dried composition forproducing galacto-oligosaccharides to be part of a product selected fromthe group consisting of yoghurt, cheese, fermented dairy products,dietary supplements and probiotic comestible products, is provided. Inone aspect, the product is yoghurt, cheese, or fermented dairy products.In one aspect, the use of a spray-dried composition as disclosed herein,for producing galacto-oligosaccharides to enhance the growth ofBifidobacterium, is provided. In one aspect, the use of a spray-driedcomposition as disclosed herein or a cell as disclosed herein, forproducing galacto-oligosaccharides to enhance the growth ofBifidobacterium in a mixed culture fermentation, is provided.

The treatment of milk products with enzymes that converts lactose intomonosaccharides or GOS have several advantages. First the products canbe consumed by people with lactose intolerance that would otherwiseexhibit symptoms such as flatulence and diarrhea. Secondly, dairyproducts treated with lactase will have a higher sweetness than similaruntreated products due to the higher perceived sweetness of glucose andgalactose compared to lactose. This effect is particularly interestingfor applications such as yoghurt and ice-cream where high sweetness ofthe end product is desired and this allows for a net reduction ofcarbohydrates in the consumed product. Thirdly, in ice-cream productiona phenomenon termed sandiness is often seen, where the lactose moleculescrystallizes due to the relative low solubility of the lactose. Whenlactose is converted into monosaccharides or GOS the mouth feeling ofthe ice-cream is much improved over the non-treated products. Thepresence of a sandy feeling due to lactose crystallization can beeliminated and the raw material costs can be decreased by replacement ofskimmed milk powder by whey powder. The main effects of the enzymatictreatment were increased sweetness.

In one aspect, the spray-dried composition as disclosed herein may beused together with other enzymes such as proteases such as chymosin orrennin, lipases such as phospholipases, amylases, transferases, andlactases. In one aspect, the transgalactosylating polypeptide(s) asdisclosed herein may be used together with lactase. This may especiallybe useful when there is a desire to reduce residual lactose aftertreatment with the transgalactosylating polypeptide(s) as disclosedherein especially at low lactose levels. A lactase in the context of thepresent invention is any glycoside hydrolase having the ability tohydrolyse the disaccharide lactose into constituent galactose andglucose monomers. The group of lactases comprises but is not limited toenzymes assigned to subclass EC 3.2.1.108. Enzymes assigned to othersubclasses, such as, e.g., EC 3.2.1.23, may also be lactases in thecontext of the present invention. A lactase in the context of theinvention may have other activities than the lactose hydrolysingactivity, such as for example a transgalactosylating activity. In thecontext of the invention, the lactose hydrolysing activity of thelactase may be referred to as its lactase activity or itsbeta-galactosidase activity. Enzymes having lactase activity to be usedin a method of the present invention may be of animal, of plant or ofmicrobial origin. Preferred enzymes are obtained from microbial sources,in particular from a filamentous fungus or yeast, or from a bacterium.The enzyme may, e.g., be derived from a strain of Agaricus, e.g. A.bisporus; Ascovaginospora; Aspergillus, e.g. A. niger, A. awamori, A.foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia;Dictyostelium, e.g. D. discoideum; Kluveromyces, e.g. K. fragilis, K.lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus;Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g.R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S.libertiana; Torula; Torulopsis; Trichophyton, e.g. T. rubrum;Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. B. coagulans, B.circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B.stearothermophilus, B. thuringiensis; Bifidobacterium, e.g. B. longum,B. bifidum, B. animalis; Chryseobacterium; Citrobacter, e.g. C.freundii; Clostridium, e.g. C. perfringens; Diplodia, e.g. D. gossypina;Enterobacter, e.g. E. aerogenes, E. cloacae Edwardsiella, E. tarda;Erwinia, e.g. E. herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g.K. pneumoniae; Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N.crassa; Proteus, e.g. P. vulgaris; Providencia, e.g. P. stuartii;Pycnoporus, e.g. Pycnoporus cinnabarinus, Pycnoporus sanguineus;Ruminococcus, e.g. R. torques; Salmonella, e.g. S. typhimurium;Serratia, e.g. S. liquefasciens, S. marcescens; Shigella, e.g. S.flexneri; Streptomyces, e.g. S. antibioticus, S. castaneoglobisporus, S.violeceoruber; Trametes; Trichoderma, e.g. T. reesei, T. viride;Yersinia, e.g. Y. enterocolitica. In one embodiment, the lactase is anintracellular component of microorganisms like Kluyveromyces andBacillus. Kluyveromyces, especially K. fragilis and K. lactis, and otherfungi such as those of the genera Candida, Torula and Torulopsis, are acommon source of fungal lactases, whereas B. coagulans and B. circulansare well known sources for bacterial lactases. Several commerciallactase preparations derived from these organisms are available such asLactozym® (available from Novozymes, Denmark), HA-Lactase (availablefrom Chr. Hansen, Denmark) and Maxilact® (available from DSM, theNetherlands), all from K. lactis. All these lactases are so calledneutral lactases having a pH optimum between pH 6 and pH 8. When suchlactases are used in the production of, e.g., low-lactose yoghurt, theenzyme treatment will either have to be done in a separate step beforefermentation or rather high enzyme dosages have to be used, becausetheir activity drop as the pH decreases during fermentation. Also, theselactases are not suitable for hydrolysis of lactose in milk performed athigh temperature, which would in some cases be beneficial in order tokeep the microbial count low and thus ensure good milk quality.

In one embodiment, the enzyme is a lactase from a bacterium, e.g. fromthe family Bifidobacteriaceae, such as from the genus Bifidobacteriumsuch as the lactase described in, inter alia, WO 2009/071539 andWO2013/182686.

Materials and Methods

Method 1

Production of Polypeptide

Synthetic genes designed to encode the Bifidobacterium bifidum fulllength (1752 residues) gene with codons optimised for expression inBacillus subtilis were purchased from GeneART (Regensburg, Germany) SEQID No. 8.

The Bifidobacterium bifidum truncation mutants were constructed usingpolymerase chain reaction with reverse primers that allowed specificamplification of the selected region of the synthetic gene.

Forward primer: (SEQ ID NO: 15)GGGGTAACTAGTGGAAGATGCAACAAGAAG (SpeI underlined).

The SEQ IDs for the truncation mutants and corresponding reverse primersare indicated in Table 2 below.

TABLE 2 Truncation mutant Primer sequence BIF917GCGCTTAATTAATTATGTTTTTTCTGTGCTTG (SEQ ID NO: 9) TTC SEQ ID NO: 16 BIF995GCGCTTAATTAATTACAGTGCGCCAATTTCAT (SEQ ID NO: 10) CAATCA SEQ ID NO: 17BIF1068 GCGCTTAATTAATTATTGAACTCTAATTGTCG (SEQ ID NO: 11)CTG SEQ ID NO: 18 BIF1172 (SEQ ID NO: 12) BIF1241GCGCTTAATTAATTATGTCGCTGTTTTCAGTT (SEQ ID NO: 13) CAAT SEQ ID NO: 19BIF1326 GCGCTTAATTAATTAAAATTCTTGTTCTGTGC (SEQ ID NO: 14)CCA SEQ ID NO: 20 BIF 1478 GCGCTTAATTAATTATCTCAGTCTAATTTCGCTTGCGC SEQ ID NO: 21

The synthetic gene was cloned into the pBNspe Bacillus subtilisexpression vector using the unique restriction sites SpeI and PacI(FIG. 1) and the isolated plasmids were transformed into a Bacillussubtilis strain. Transformants were restreaked onto LB plates containing10 μg/mL Neomycin as selection.

A preculture was setup in LB media containing 10 μg/mL Neomycin andcultivated for 7 hours at 37° C. and 180 rpm shaking. 500 μL of thispreculture was used to inoculate 50 mL Grant's modified mediumcontaining 10 μg/mL Neomycin at allowed to grow for 68 hours at 33° C.and 180 rpm shaking.

Cells were lysed by addition directly to the culture media of 1 mg/mlLysozyme (Sigma-Aldrich) and 10 U/ml Benzonase (Merck) finalconcentrations and incubated for 1 hr at 33° C. at 180 RPM. Lysates werecleared by centrifugation at 10.000×g for 20 minutes and subsequentlysterile filtered.

Grant's Modified Media was Prepared According to the FollowingDirections:

Part I (Autoclave)

Soytone 10 g Bring to 500 mL per liter

Part II

1M K₂HPO₄ 3 mL Glucose 75 g Urea 3.6 g Grant's 10X MOPS 100 mL Bring to400 mL per liter

PART I (2 w/w % Soytone) was prepared, and autoclaved for 25 minutes at121° C.

PART II was prepared, and mixed with PART 1 and pH was adjusted to pH to7.3 with HCl/NaOH.

The volume was brought to full volume and sterilized through 0.22-μm PESfilter.

10×MOPS Buffer was prepared according to the following directions:

83.72 g Tricine 7.17 g KOH Pellets 12 g NaCl 29.22 g 0.276M K2SO4 10 mL0.528M MgCl2 10 mL Grant's Micronutrients 100X

Bring to app. 900 mL with water and dissolve. Adjust pH to 7.4 with KOH,fill up to 1 L and sterile filter the solution through 0.2 μm PESfilter.

100× Micronutrients was prepared according to the following directions:

Sodium Citrate•2H2O 1.47 g CaCl2•2H2O 1.47 g FeSO4•7H2O 0.4 g MnSO4•H2O0.1 g ZnSO4•H2O 0.1 g CuCl2•2H2O 0.05 g CoCl2•6H2O 0.1 g Na2MoO4•2H2O0.1 g

Dissolve and adjust volume to 1 L with water.

Sterilization was through 0.2 μm PES filter.

Storing was at 4° C. avoid light.

Method 2

Purification and Enzyme Preparations

The filtrated enzyme isolate was concentrated using a VivaSpin ultrafiltration device with a 10 kDa MW cut off (Vivaspin 20, Sartorius,Lot#12VS2004) and the concentrate was loaded onto a PD10 desaltingcolumn (GE healthcare, Lot#6284601) and eluted in 20 mM Tris-HCl pH 8.6.Chromatography was carried out manually on an Akta FPLC system (GEHealthcare). 4 mL of the desalted sample, containing approximately 20 mgprotein, was loaded onto a 2 mL HyperQ column (HyperCel™, Q sorbent)equilibrated with 20 mM Tris-HCl pH 8.6 at a flowrate of 1 ml/min. Thecolumn was thoughroughly washed with 30 CV (column volumes) wash bufferand the bound β-galactosidase was eluted with a 100CV long gradient into20 mM Tris-HCl pH 8.6 250 mM NaCl. Remaining impurities on the columnwere removed with a one-step elution using 20 mM Tris-HCl pH 8.6 500 mMNaCl. Protein in the flow through and elution was analyzed forβ-galactosidase activity and by SDS-page.

SDS-page gels were run with the Invitrogen NuPage® Novex 4-12% Bis-Trisgel 1.0 mm, 10 well (Cat#NP0321box), See-Blue® Plus2 prestained Standard(Cat#LC5925) and NuPAGE® MES SDS Running Buffer (Cat#NP0002) accordingto the manufacturer's protocol. Gels were stained with Simply BlueSafestain (Invitrogen, Cat#LC6060) (FIG. 2).

Method 3

Measuring β-Galactosidase Activity

Enzymatic activity was measured using the commercially availablesubstrate 2-Nitrophenyl-β-D-Galactopyranoside (ONPG) (Sigma N1127).

ONPG w/o Acceptor

100 mM KPO4 pH 6.0 12.3 mM ONPG

ONPG Supplemented with Acceptor

100 mM KPO4 pH 6.0 20 mM Cellobiose 12.3 mM ONPG

STOP Solution

10% Na2CO3

10 μl dilution series of purified enzyme was added in wells of amicrotiter plates containing 90 μl ONPG-buffer with or without acceptor.Samples were mixed and incubated for 10 min at 37° C., subsequently 100μl STOP Solution were added to each well to terminate reaction.Absorbance measurements were recorded at 420 nm on a Molecular DeviceSpectraMax platereader controlled by the Softmax software package.

The ratio of transgalactosylation activity was calculated as follows:

Ratio of transgalctosylationactivity=(Abs420^(+Cellobiose)/Abs420^(−Cellobiose))*100, for dilutionswhere the absorbance was between 0.5 and 1.0 (FIG. 3).

Method 4

Determination of LAU Activity

Principle:

The principle of this assay method is that lactase hydrolyzes2-o-nitrophenyl-β-D-galactopyranoside (ONPG) into 2-o-nitrophenol (ONP)and galactose at 37° C. The reaction is stopped with the sodiumcarbonate and the liberated ONP is measured in spectrophotometer orcolorimeter at 420 nm.

Reagents:

MES buffer pH 6.4 (100 mM MES pH 6.4, 10 mM CaCl₂): Dissolve 19.52 g MEShydrate (Mw: 195.2 g/mol, Sigma-aldrich #M8250-250G) and 1.470 g CaCl₂di-hydrate (Mw: 147.01 g/mol, Sigma-aldrich) in 1000 ml ddH₂O, adjust pHto 6.4 by 10M NaOH. Filter the solution through 0.2 μm filter and storeat 4° C. up to 1 month.

ONPG substrate pH 6.4 (12.28 mM ONPG, 100 mM MES pH 6.4, 10 mM CaCl₂):Dissolve 0.370 g 2-o-nitrophenyl-β-D-galactopyranoside (ONPG, Mw: 301.55g/mol, Sigma-aldrich #N1127) in 100 ml MES buffer pH 6.4 and store darkat 4° C. for up to 7 days.

Stop reagent (10% Na2CO3): Dissolve 20.0 g Na2CO3 in 200 ml ddH₂O,Filter the solution through 0.2 μm filter and store at RT up to 1 month.

Procedure:

Dilution series of the enzyme sample was made in the MES buffer pH 6.4and 10 μL of each sample dilution were transferred to the wells of amicrotiter plate (96 well format) containing 90 μl ON PG substrate pH6.4. The samples were mixed and incubated for 5 min at 37° C. using aThermomixer (Comfort Thermomixer, Eppendorf) and subsequently 100 μlStop reagent was added to each well to terminate the reaction. A blankwas constructed using MES buffer pH 6.4 instead of the enzyme sample.The increase in absorbance at 420 nm was measured at a ELISA reader(SpectraMax platereader, Molecular Device) against the blank.

Calculation of Enzyme Activity:

The molar extinction coefficient of 2-o-nitrophenol (Sigma-aldrich#33444-25G) in MES buffer pH 6.4 was determined (0.5998×10⁻⁶ M⁻¹×cm⁻¹).One unit (U) of lactase activity (LAU) was defined as that correspondingto the hydrolysis of 1 nmol of ON PG per minute. Using microtitre plateswith a total reaction volume of 200 μL (light path of 0.52 cm) thelactase activity per mL of the enzyme sample may be calculated using thefollowing equation:

${{{LAU}/{ml}}\mspace{11mu} \left( \frac{nmol}{\min \cdot {mL}} \right)} = \frac{{Abs}_{420} \times 200\mspace{14mu} {µL} \times {dilution}\mspace{14mu} {factor}}{\begin{matrix}{{0.5998` \cdot 10^{3} \cdot {nM}^{- 1} \cdot {cm}^{- 1}} \times} \\{0.52\mspace{11mu} {cm} \times 5\mspace{14mu} \min \times 0.01\mspace{14mu} {mL}}\end{matrix}}$

Calculation of Specific Activity for BIF917 Shown Herein as SEQ ID NO:1:

Determination of BIF917 Concentration:

Quantification of the target enzyme (BIF917) and truncation productswere determined using the Criterion Stain free SDS-page system (BioRad).Any kD Stain free precast Gel 4-20% Tris-HCl, 18 well (Comb #345-0418)was used with a Serva Tris-Glycine/SDS buffer (BioRad cat. #42529). Gelswere run with the following parameters: 200 V, 120 mA, 25 W, 50 min. BSA(1.43 mg/ml) (Sigma-Aldrich, cat. #500-0007) was used as proteinstandard and Criterion Stain Free Imager (BioRad) was used with ImageLab software (BioRad) for quantification using band intensity withcorrelation of the tryptophan content.

The specific LAU activity of BIF917 was determined from crude ferment(ultra filtration concentrate) of two independent fermentations (asdescribed in method 1) and using 5 different dilutions (see table 1).

The specific activity of BIF917 was found to be 21.3 LAU/mg or 0.0213LAU/ppm.

TABLE 1 Determination of BIF917 specific activity Protein Protein(BIF917) (BIF917) Specific Specific Dilution Activity concentrationconcentration activity activity Sample ID Enzyme Fermentation factorLAU/ml mg/ml ppm LAU/mg LAU/ppm 1 BIF 917 a 5 26.9 1.23 1232 21.9 0.02192 BIF 917 a 10 53.9 2.44 2437 22.1 0.0221 3 BIF 917 a 10 75.4 3.56 355621.2 0.0212 4 BIF 917 a 20 163.9 7.78 7778 21.1 0.0211 5 BIF 917 a 30233.6 11.06 11065 21.1 0.0211 6 BIF 917 b 5 30.26825 1.34 1342 22.60.0226 7 BIF 917 b 10 55.91536 2.61 2607 21.4 0.0214 8 BIF 917 b 1076.96056 3.70 3697 20.8 0.0208 9 BIF 917 b 20 156.986 7.75 7755 20.20.0202 10 BIF 917 b 30 236.9734 11.45 11452 20.7 0.0207 Arg 21.3 0.0213Std 0.700976 0.000701

Example 1

An experiment was conducted to test the effects of diafiltration (DF),bentionite treatment (BT) and heat treatment (HT) on the physicalstability of BIF917 formulated with glycerol. Formulation details aregiven in the Table below:

Formulation Sample1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample7 Sample 8 Expected U/g 1370 685 685 685 685 685 685 685 ActivityMeasured U/g 1012 863 1016 847 1005 897 930 850 Activity Cosolvent —0.00% 49.70% 49.70% 49.70% 49.80% 49.80% 49.80% 49.80% w/w NaCl — 0.00%0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% w/w preservative — 0.14% 0.14%0.14% 0.14% 0.14% 0.14% 0.14% 0.14% w/w UFC — 99.37% 49.71% 49.68%49.66% 49.83% 49.83% 49.81% 49.83% w/w Water — 0.00% 0.00% 0.00% 0.00%0.00% 0.00% 0.00% 0.00% w/w

Akt Akt X dilut. F X dilut. F Nmol/ Nmol/ −cello- +cello- −cello-+cello- min. min Time = Dilut. biose biose biose biose Ratio −cello-+cello- nr Enzyme id: “variant” Info X days Factor Avg-bl Avg-bl Avg-blAvg-bl % brose biose 1 MW Stability Control BIF_917 0 200 0.395 1.09378.980 218.500 277 1012.9 2802.2 #3 - sample 1 2 MW Stability HT 0 2000.336 0.988 67.290 197.630 294 863.0 2534.5 #3 - sample 2 3 MW StabilityBT 0 200 0.396 1.134 79.230 226.820 286 1016.1 2908.9 #3 - sample 3 4 MWStability BT + HT 0 200 0.330 0.983 66.020 196.610 298 846.7 2521.4 #3 -sample 4 5 MW Stability DF 0 200 0.392 1.120 78.380 223.940 286 1005.22871.9 #3 - sample 5 6 MW Stability DF + HT 0 200 0.350 1.013 69.930202.610 290 896.8 2598.4 #3 - sample 6 7 MW Stability DF + BT 0 2000.362 1.057 72.480 211.300 292 929.5 2709.8 #3 - sample 7 8 MW StabilityDF + BT + 0 200 0.332 0.922 66.310 184.400 278 850.4 2364.9 #3 - sample8 HT

The Samples were incubated at each of the following storage conditions,5° C., 20° C. and 37° C.

The results are shown in FIG. 1.

Example 2

The effect of the formulation on actual GOS forming enzymatic activityon the application relevant substrate, lactose, was also investigated.It was surprisingly found that the presence of glycerol in theapplication had significant negative effect on the GOS generationactivity due to generation of the undesired galactosyl-glycerol ratherthan the desired GOS in the application. The presence of thegalactosyl-glycerol can only be detected when the actual reactionproducts are analysed by for example HPLC and not by the chromogenicsubstrate ONPG since this assay only measure the release of ONP. Inparticular it was surprisingly found (as illustrated in FIGS. 2 and 3)that the use of greater than 0.1 wt % of glycerol perturbs thetransgalactosylation reaction resulting in lower yields of the desiredGOS. The low amounts of glycerol need to perturb the GOS generationindicates that the enzyme is an efficient galactosyl-glycerol generatingenzyme in the presence of free glycerol.

Example 3

The BIF917 enzyme was mixed to provide an intermediate formulationaccording to the following Table and subsequently spray-dried.

Composition Name #1 #2 #3 #4 #5 Spray Feed preparation Target ActualActual Actual Actual Actual value value value value value value 10% NaClkg 5.000 pH = 5, carbon treatment, 0.4% kg 5.000 sorbate + 10% NaCl 20%NaCl kg 5.000 Lead sorbate kg 5.000 Lead sorbate kg 5.000 MD DE20 kg1.000 1.000 1.000 1.000 2.000 Composition 10% NaCl % 83.3% 0.0% 0.0%0.0% 0.0% pH = 5, carbon treatment, 0.4% % 0.0% 83.3% 0.0% 0.0% 0.0%sorbate + 10% NaCl Lead 20% NaCl % 0.0% 0.0% 83.3% 0.0% 0.0% Lead sorb %0.0% 0.0% 0.0% 83.3% 0.0% Lead sorb % 0.0% 0.0% 0.0% 0.0% 71.4% MD DE20% 16.7% 16.7% 16.7% 16.7% 28.6% Mix temp ° C. Mix time before spray min<30 min <30 min <30 min <30 min <30 min #1 #2 #3 #4 #5 Spray DryingActual Actual Actual Actual Actual value value value value value Spraytime min 9.5 10 9 11.5 11 Inlet air temp (before 0° C. outdoor outdooroutdoor outdoor outdoor heating): Process air temp inlet 165° C. 175 170172 172 174 Process air temp outlet 85° C. 93-86 85-81 87-86 85-81 84-79Process air flow 600 m3/hr Atomizer wheel Ø120 mm 12000 rpm 20000 2000020000 20000 20000 Feed rate (kg dispersion/hr) 37.9 36.0 40.0 31.3 38.2WM505U pump/8 mm ID Ø rpm 127-135 127-125 155 108-106 155 tubing) Sample1.089 1.22 1.67 1.089 1.747 sample B-grade up/down 0.296 0.656 0.7830.296 0.635 Yield 61.6% 83.4% 89.2% 79.1% 86.6% DM in enzyme rawmaterial 25.0% 25.0% 35.0% 15.0% 15.0% Powder temparature °C. >43 >41 >48 >44 >49

The effect of the formulation on activity was investigated at each ofthe following storage conditions: 5° C., 20° C. and 3TC. The results forsample #1, 3 and 5 are shown in FIG. 4 and they show that goodtransgalactosylation activity is retained following formulation.

Example 4

The effect of potato starch on dust reduction is illustrated in FIG. 5.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in chemistry, biochemistry, biology, or related fields areintended to be within the scope of the following claims.

List of sequences >SEQ ID NO: 1 (BIF_917)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfsingekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrytfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstekt >SEQ ID NO: 2 (BIF_995)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrytfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdaysddqiakagsfsvagtvagqkisvrytmideigal >SEQ ID NO: 3 (BIF_1068)Vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrytfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdaysddqiakagsfsvagtvagqkisvrytmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvq >SEQ ID NO: 4 (BIF_1172)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdaysddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssysgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqi >SEQ ID NO: 5 (BIF_1241)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdaysddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssysgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqisadgknwtdlaatetiaaqessdrvkpytydfapvgatfvkvtvtnadtttpsgvvcaglteielktat >SEQ ID NO: 6 (BIF_1326)vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdaysddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssysgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqisadgknwtdlaatetiaaqessdrvkpytydfapvgatfvkvtvtnadtttpsgvvcaglteielktatskfvtntsaalssltvngtkvsdsvlaagsyntpaiiadvkaegegnasvtvlpandnvirvitesedhvtrktftinlgteqef>SEQ ID NO: 7 Bifidobacterium bifidum glycoside hydrolase catalytic coreqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrIpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnIttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevInggkvIdtydteygfrwtgfdatsgfsIngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvIvveevfdmwnrskngntedygkwfgqaiagdnavIggdkdetwakfdltstinrdrnapsvimwsIgnemmegisgsysgfpatsakIvawtkaadstrpmty >SEQ ID NO: 8 nucleotide sequence encoding full lengthgcagttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagthttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagthttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtattthttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatcagcgcagatggcaaaaattggacagatctggcagcaacagaaacaattgcagcgcaagaatcaagcgatagagtcaaaccgtatacatatgattttgcaccggttggcgcaacatttgttaaagtgacagtcacaaacgcagatacaacaacaccgtcaggcgttgtttgcgcaggcctgacagaaattgaactgaaaacagcgacaagcaaatttgtcacaaatacatcagcagcactgtcatcacttacagtcaatggcacaaaagtttcagattcagttctggcagcaggctcatataacacaccggcaattatcgcagatgttaaagcggaaggcgaaggcaatgcaagcgttacagtccttccggcacatgataatgttattcgcgtcattacagaaagcgaagatcatgtcacacgcaaaacatttacaatcaacctgggcacagaacaagaatttccggctgattcagatgaaagagattatccggcagcagatatgacagtcacagttggctcagaacaaacatcaggcacagcaacagaaggaccgaaaaaatttgcagtcgatggcaacacatcaacatattggcatagcaattggacaccgacaacagttaatgatctgtggatcgcgtttgaactgcaaaaaccgacaaaactggatgcactgagatatcttccgcgtccggcaggctcaaaaaatggcagcgtcacagaatataaagttcaggtgtcagatgatggaacaaactggacagatgcaggctcaggcacatggacaacggattatggctggaaactggcggaatttaatcaaccggtcacaacaaaacatgttagactgaaagcggttcatacatatgcagatagcggcaacgataaatttatgagcgcaagcgaaattagactgagaaaagcggtcgatacaacggatatttcaggcgcaacagttacagttccggcaaaactgacagttgatagagttgatgcagatcatccggcaacatttgcaacaaaagatgtcacagttacactgggagatgcaacactgagatatggcgttgattatctgctggattatgcaggcaatacagcagttggcaaagcaacagtgacagttagaggcattgataaatattcaggcacagtcgcgaaaacatttacaattgaactgaaaaatgcaccggcaccggaaccgacactgacatcagttagcgtcaaaacaaaaccgagcaaactgacatatgttgtcggagatgcatttgatccggcaggcctggttctgcaacatgatagacaagcagatagacctccgcaaccgctggttggcgaacaagcggatgaacgcggactgacatgcggcacaagatgcgatagagttgaacaactgcgcaaacatgaaaatagagaagcgcatagaacaggcctggatcatctggaatttgttggcgcagcagatggcgcagttggagaacaagcaacatttaaagtccatgtccatgcagatcagggagatggcagacatgatgatgcagatgaacgcgatattgatccgcatgttccggtcgatcatgcagttggcgaactggcaagagcagcatgccatcatgttattggcctgagagtcgatacacatagacttaaagcaagcggctttcaaattccggctgatgatatggcagaaatcgatcgcattacaggctttcatcgttttgaacgccatgtc >SEQ ID NO: 9 nucleotide sequence encoding BIF_917gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaaca >SEQ ID NO: 10 nucleotide sequence encoding BIF_995gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactg >SEQ ID NO: 11 nucleotide sequence encoding BIF_1068gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagthttggcaaagaatttaaagtcacagcgacaattagagttcaa >SEQ ID NO: 12 nucleotide sequence encoding BIF_1172gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagthttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatc >SEQ ID NO: 13 nucleotide sequence encoding BIF_1241gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagthttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatcagcgcagatggcaaaaattggacagatctggcagcaacagaaacaattgcagcgcaagaatcaagcgatagagtcaaaccgtatacatatgattttgcaccggttggcgcaacatttgttaaagtgacagtcacaaacgcagatacaacaacaccgtcaggcgttgthgcgcaggcctgacagaaattgaactgaaaacagcgaca >SEQ ID NO: 14 nucleotide sequence encoding BIF_1326gttgaagatgcaacaagaagcgatagcacaacacaaatgtcatcaacaccggaagttgtttattcatcagcggtcgatagcaaacaaaatcgcacaagcgattttgatgcgaactggaaatttatgctgtcagatagcgttcaagcacaagatccggcatttgatgattcagcatggcaacaagttgatctgccgcatgattatagcatcacacagaaatatagccaaagcaatgaagcagaatcagcatatcttccgggaggcacaggctggtatagaaaaagctttacaattgatagagatctggcaggcaaacgcattgcgattaattttgatggcgtctatatgaatgcaacagtctggtttaatggcgttaaactgggcacacatccgtatggctattcaccgttttcatttgatctgacaggcaatgcaaaatttggcggagaaaacacaattgtcgtcaaagttgaaaatagactgccgtcatcaagatggtattcaggcagcggcatttatagagatgttacactgacagttacagatggcgttcatgttggcaataatggcgtcgcaattaaaacaccgtcactggcaacacaaaatggcggagatgtcacaatgaacctgacaacaaaagtcgcgaatgatacagaagcagcagcgaacattacactgaaacagacagtttttccgaaaggcggaaaaacggatgcagcaattggcacagttacaacagcatcaaaatcaattgcagcaggcgcatcagcagatgttacaagcacaattacagcagcaagcccgaaactgtggtcaattaaaaacccgaacctgtatacagttagaacagaagttctgaacggaggcaaagttctggatacatatgatacagaatatggctttcgctggacaggctttgatgcaacatcaggcttttcactgaatggcgaaaaagtcaaactgaaaggcgttagcatgcatcatgatcaaggctcacttggcgcagttgcaaatagacgcgcaattgaaagacaagtcgaaatcctgcaaaaaatgggcgtcaatagcattcgcacaacacataatccggcagcaaaagcactgattgatgtctgcaatgaaaaaggcgttctggttgtcgaagaagtctttgatatgtggaaccgcagcaaaaatggcaacacggaagattatggcaaatggtttggccaagcaattgcaggcgataatgcagttctgggaggcgataaagatgaaacatgggcgaaatttgatcttacatcaacaattaaccgcgatagaaatgcaccgtcagttattatgtggtcactgggcaatgaaatgatggaaggcatttcaggctcagtttcaggctttccggcaacatcagcaaaactggttgcatggacaaaagcagcagattcaacaagaccgatgacatatggcgataacaaaattaaagcgaactggaacgaatcaaatacaatgggcgataatctgacagcaaatggcggagttgttggcacaaattattcagatggcgcaaactatgataaaattcgtacaacacatccgtcatgggcaatttatggctcagaaacagcatcagcgattaatagccgtggcatttataatagaacaacaggcggagcacaatcatcagataaacagctgacaagctatgataattcagcagttggctggggagcagttgcatcatcagcatggtatgatgttgttcagagagattttgtcgcaggcacatatgtttggacaggatttgattatctgggcgaaccgacaccgtggaatggcacaggctcaggcgcagttggctcatggccgtcaccgaaaaatagctattttggcatcgttgatacagcaggctttccgaaagatacatattatttttatcagagccagtggaatgatgatgttcatacactgcatattcttccggcatggaatgaaaatgttgttgcaaaaggctcaggcaataatgttccggttgtcgtttatacagatgcagcgaaagtgaaactgtattttacaccgaaaggctcaacagaaaaaagactgatcggcgaaaaatcatttacaaaaaaaacaacagcggcaggctatacatatcaagtctatgaaggcagcgataaagattcaacagcgcataaaaacatgtatctgacatggaatgttccgtgggcagaaggcacaatttcagcggaagcgtatgatgaaaataatcgcctgattccggaaggcagcacagaaggcaacgcatcagttacaacaacaggcaaagcagcaaaactgaaagcagatgcggatcgcaaaacaattacagcggatggcaaagatctgtcatatattgaagtcgatgtcacagatgcaaatggccatattgttccggatgcagcaaatagagtcacatttgatgttaaaggcgcaggcaaactggttggcgttgataatggctcatcaccggatcatgattcatatcaagcggataaccgcaaagcattttcaggcaaagtcctggcaattgttcagtcaacaaaagaagcaggcgaaattacagttacagcaaaagcagatggcctgcaatcaagcacagttaaaattgcaacaacagcagttccgggaacaagcacagaaaaaacagtccgcagcttttattacagccgcaactattatgtcaaaacaggcaacaaaccgattctgccgtcagatgttgaagttcgctattcagatggaacaagcgatagacaaaacgttacatgggatgcagtttcagatgatcaaattgcaaaagcaggctcattttcagttgcaggcacagttgcaggccaaaaaattagcgttcgcgtcacaatgattgatgaaattggcgcactgctgaattattcagcaagcacaccggttggcacaccggcagttcttccgggatcaagaccggcagtcctgccggatggcacagtcacatcagcaaattttgcagtccattggacaaaaccggcagatacagtctataatacagcaggcacagtcaaagtaccgggaacagcaacagthttggcaaagaatttaaagtcacagcgacaattagagttcaaagaagccaagttacaattggctcatcagtttcaggaaatgcactgagactgacacaaaatattccggcagataaacaatcagatacactggatgcgattaaagatggctcaacaacagttgatgcaaatacaggcggaggcgcaaatccgtcagcatggacaaattgggcatattcaaaagcaggccataacacagcggaaattacatttgaatatgcgacagaacaacaactgggccagatcgtcatgtatttttttcgcgatagcaatgcagttagatttccggatgctggcaaaacaaaaattcagatcagcgcagatggcaaaaattggacagatctggcagcaacagaaacaattgcagcgcaagaatcaagcgatagagtcaaaccgtatacatatgattttgcaccggttggcgcaacatttgttaaagtgacagtcacaaacgcagatacaacaacaccgtcaggcgttgthgcgcaggcctgacagaaattgaactgaaaacagcgacaagcaaatttgtcacaaatacatcagcagcactgtcatcacttacagtcaatggcacaaaagtttcagattcagttctggcagcaggctcatataacacaccggcaattatcgcagatgttaaagcggaaggcgaaggcaatgcaagcgttacagtccttccggcacatgataatgttattcgcgtcattacagaaagcgaagatcatgtcacacgcaaaacatttacaatcaacctgggcacagaacaagaattt >SEQ ID NO: 15 forward primer for generation of BIF variantsGGGGTAACTAGTGGAAGATGCAACAAGAAG >SEQ ID NO: 16 reverse primer for BIF917GCGCTTAATTAATTATGTTTTTTCTGTGCTTGTTC >SEQ ID NO: 17 reverse primer for BIF995GCGCTTAATTAATTACAGTGCGCCAATTTCATCAATCA >SEQ ID NO: 18 reverse primer for BIF1068GCGCTTAATTAATTATTGAACTCTAATTGTCGCTG >SEQ ID NO: 19 reverse primer for BIF1241GCGCTTAATTAATTATGTCGCTGTTTTCAGTTCAAT >SEQ ID NO: 20 reverse primer for BIF1326GCGCTTAATTAATTAAAATTCTTGTTCTGTGCCCA >SEQ ID NO: 21 reverse primer for BIF1478GCGCTTAATTAATTATCTCAGTCTAATTTCGCTTGCGC>SEQ ID NO: 22 Bifidobacterium bifidum BIF1750vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdysitqkysqsneaesaylpggtgwyrksftidrdlagkriainfdgvymnatvwfngvklgthpygyspfsfdltgnakfggentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslatqnggdytmnlttkvandteaaanitlkqtvfpkggktdaaigtvttasksiaagasadvtstitaaspklwsiknpnlytvrtevlnggkvldtydteygfrwtgfdatsgfslngekvklkgvsmhhdqgslgavanrraierqveilqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwnrskngntedygkwfgqaiagdnavlggdkdetwakfdltstinrdrnapsvimwslgnemmegisgsysgfpatsaklvawtkaadstrpmtygdnkikanwnesntmgdnltanggvvgtnysdganydkirtthpswaiygsetasainsrgiynrttggaqssdkqltsydnsavgwgavassawydvvqrdfvagtyvwtgfdylgeptpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqsqwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvklyftpkgstekrligeksftkkttaagytyqvyegsdkdstahknmyltwnvpwaegtisaeaydennrlipegstegnasytttgkaaklkadadrktitadgkdlsyievdvtdanghivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrkafsgkvlaivqstkeageitvtakadglqsstvkiattavpgtstektvrsfyysrnyyvktgnkpilpsdvevrysdgtsdrqnvtwdaysddqiakagsfsvagtvagqkisvrvtmideigallnysastpvgtpavlpgsrpavlpdgtvtsanfavhwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrsqvtigssysgnalrltqnipadkqsdtldaikdgsttvdantggganpsawtnwayskaghntaeitfeyateqqlgqivmyffrdsnavrfpdagktkiqisadgknwtdlaatetiaaqessdrvkpytydfapvgatfvkvtvtnadtttpsgvvcaglteielktatskfvtntsaalssltvngtkvsdsvlaagsyntpaiiadvkaegegnasvtvlpandnvirvitesedhvtrktftinlgteqefpadsderdypaadmtvtvgseqtsgtategpkkfavdgntstywhsnwtpttyndlwiafelqkptkldalrylprpagskngsvteykvqvsddgtnwtdagsgtwttdygwklaefnqpvttkhvrlkavhtyadsgndkfmsaseirlrkavdttdisgatvtvpakltvdrvdadhpatfatkdvtvtlgdatlrygvdylldyagntavgkatvtvrgidkysgtvaktftielknapapeptltsysyktkpskltyvvgdafdpaglvlqhdrqadrppqplvgeqadergltcgtrcdrveqlrkhenreahrtgldhlefvgaadgavgeqatfkvhvhadqgdgrhddaderdidphypvdhavgelaraachhviglrvdthrlkasgfqipaddmaeidritgfhrferhvg>SEQ ID NO: 23 The signal sequence of extracellular lactase from Bifidobacteriumbifidum DSM20215 Vrskklwisllfalaliftmafgstssaqa

1. A spray-dried composition comprising a polypeptide which is aβ-galactosidase having transgalactosylating activity and a maltodextrin.2. A spray-dried composition comprising a polypeptide which is aβ-galactosidase having transgalactosylating activity and sodiumchloride.
 3. The spray-dried composition according to claim 1 or claim 2comprising a polypeptide which is a β-galactosidase havingtransgalactosylating activity, a maltodextrin and sodium chloride. 4.The composition of any preceding claim, wherein the polypeptide is anenzyme which is classified in Enzyme Classification (E.C.) 3.2.1.23. 5.The composition of any preceding claim, wherein the polypeptide has aratio of transgalactosylating activity: β-galactosidase activity of atleast 0.5, at least 1, at least 2, at least 2.5, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, or at least 12 at or above a concentration of 3% w/winitial lactose concentration.
 6. The composition of any one of claims 1to 4, wherein the polypeptide has a transgalactosylating activity suchthat more than 20%, more than 30%, more than 40%, up to 50% of theinitial lactose is transgalactosylated as measured at a concentration of100 ppm in a milk-based assay at 37° C. and 5 w/w % lactose after 15, 30or 180 such as 180 minutes of reaction.
 7. The composition of any one ofclaims 1 to 4, wherein the polypeptide has a β-galactosidase activitysuch that less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20% of the lactose has beenhydrolysed as measured at a concentration of 100 ppm in a milk-basedassay at 37° C. and 5 w/w % lactose after 15, 30 or 180 such as 180minutes of reaction.
 8. The composition of any one of claims 5 to 7wherein the activity is retained for a period of at least 1, at least 2,at least 3, at least 4, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 18, or at least 24months.
 9. The composition of any preceding claim, wherein thepolypeptide having a transgalactosylating activity is selected from thegroup consisting of: a. a polypeptide comprising an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 1, wherein saidpolypeptide consists of at most 980 amino acid residues, b. apolypeptide comprising an amino acid sequence having at least 97%sequence identity with SEQ ID NO: 2, wherein said polypeptide consistsof at most 975 amino acid residues, c. a polypeptide comprising an aminoacid sequence having at least 96.5% sequence identity with SEQ ID NO: 3,wherein said polypeptide consists of at most 1300 amino acid residues,d. a polypeptide encoded by a polynucleotide that hybridizes under atleast low stringency conditions with i) the nucleic acid sequencecomprised in SEQ ID NO: 9, 10, 11, 12 or 13 encoding the polypeptide ofSEQ ID NO: 1, 2, 3, 4 or 5; or ii) the complementary strand of i), e. apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 70% identity to the nucleotide sequence encoding for thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5 or the nucleotide sequencecomprised in SEQ ID NO: 9, 10, 11, 12 or 13 encoding a maturepolypeptide, and f. a polypeptide comprising a deletion, insertionand/or conservative substitution of one or more amino acid residues ofSEQ ID NO: 1, 2, 3, 4 or
 5. 10. The composition according to anypreceding claim wherein the polypeptide having transgalactosylatingactivity is selected from the group consisting of: a. a polypeptidecomprising an amino acid sequence having at least 96.5% sequenceidentity with SEQ ID NO: 3, wherein said polypeptide consists of at most1300 amino acid residues, b. a polypeptide comprising an amino acidsequence having at least 90% sequence identity with SEQ ID NO: 1,wherein said polypeptide consists of at most 980 amino acid residues, c.a polypeptide encoded by a polynucleotide that hybridizes under at leastlow stringency conditions with i) the nucleic acid sequence comprised inSEQ ID NO: 9, 10, 11, 12 or 13 encoding the polypeptide of SEQ ID NO: 1,2, 3, 4, or 5; or ii) the complementary strand of i), d. a polypeptideencoded by a polynucleotide comprising a nucleotide sequence having atleast 70% identity to the nucleotide sequence encoding for thepolypeptide of SEQ ID NO: 1, 2, 3, 4 or 5 or the nucleotide sequencecomprised in SEQ ID NO: 9, 10, 11, 12 or 13 encoding a maturepolypeptide, and e. a polypeptide comprising a deletion, insertionand/or conservative substitution of one or more amino acid residues ofSEQ ID NO: 1, 2, 3, 4 or
 5. 11. The composition according to anypreceding claim, wherein the polypeptide having transgalactosylatingactivity comprises or consists of the amino acid sequence of SEQ IDNO:1, 2, 3, 4 or
 5. 12. The composition according to any precedingclaim, wherein the composition contains 0.1 wt % or less polyol.
 13. Thecomposition according to any preceding claim, wherein the compositionfurther contains potato starch.
 14. A method of spray drying acomposition comprising: a. introducing a composition into a spray dryingapparatus, wherein the composition comprises an enzyme as defined in anyone of claims 1 to 13 and a maltodextrin; and b. spray drying thecomposition to produce particles.
 15. A method of spray drying acomposition comprising: a. introducing a composition into a spray dryingapparatus, wherein the composition comprises an enzyme as defined in anyone of claims 1 to 13 and sodium chloride; and b. spray drying thecomposition to produce particles.
 16. A method for producing a foodproduct by treating a substrate comprising lactose with a composition asdefined in any one of claims 1 to
 13. 17. The method according to claim15 for producing a dairy product by treating a milk-based substratecomprising lactose with a composition as defined in any one of claims 1to
 13. 18. A process for producing galacto-oligosaccharides, comprisingcontacting the composition of any one of claims 1 to 13 with amilk-based solution comprising lactose.